Activatable binding polypeptides and methods of identification and use thereof

ABSTRACT

Activatable binding polypeptides (ABPs), which contain a target binding moiety (TBM), a masking moiety (MM), and a cleavable moiety (CM) are provided. Activatable antibody compositions, which contain a TBM containing an antigen binding domain (ABD), a MM and a CM are provided. Furthermore, ABPs which contain a first TBM, a second TBM and a CM are provided. The ABPs exhibit an “activatable” conformation such that at least one of the TBMs is less accessible to target when uncleaved than after cleavage of the CM in the presence of a cleaving agent capable of cleaving the CM. Further provided are libraries of candidate ABPs, methods of screening to identify such ABPs, and methods of use. Further provided are ABPs having TBMs that bind VEGF, CTLA-4, or VCAM, ABPs having a first TBM that binds VEGF and a second TBM that binds FGF, as well as compositions and methods of use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 13/413,447,filed on Mar. 6, 2012, now U.S. Pat. No. 8,529,898, which application isa continuation of application Ser. No. 12/196,269, filed on Aug. 21,2008, now abandoned, which application claims the benefit of U.S.Provisional Patent Application Nos. 60/957,449 filed Aug. 22, 2007;60/957,453, filed Aug. 22, 2007; and 61/052,986, filed May 13, 2008,which applications are incorporated herein by reference in theirentirety and for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Federal Grant Nos.1 U54 CA119335-01 and R43CA132498-01A1 awarded by the NationalInstitutes of Health. The Government has certain rights in thisinvention.

BACKGROUND

Protein drugs have changed the face of modern medicine, findingapplication in a variety of different diseases such a cancer, anemia,and neutropenia. As with any drugs, however, the need and desire fordrugs having improved specificity and selectivity for their targets isof great interest, especially in developing second generation of proteindrugs having known targets to which they bind.

In the realm of small molecule drugs, strategies have been developed toprovide “prodrugs” of an active chemical entity. Such prodrugs areadministered in a relatively inactive (or significantly less active)form. Once administered, the prodrug is metabolized in vivo into theactive compound. Such prodrug strategies can provide for increasedselectivity of the drug for its intended target. An example of this canbe seen in many anti-cancer treatments, in which the reduction ofadverse effects is always of paramount importance. Drugs used to targethypoxic cancer cells, through the use of redox-activation, utilize thelarge quantities of reductase enzyme present in the hypoxic cell toconvert the drug into its cytotoxic form, essentially activating it.Since the prodrug has low cytotoxicity prior to this activation, thereis a markedly decreased risk of damage to non-cancerous cells, therebyproviding for reduced side-effects associated with the drug.

There is a need in the field for a strategy for providing features of aprodrug to protein-based therapeutics.

SUMMARY OF THE INVENTION

The present disclosure provides activatable binding polypeptides (ABPs),which contain a target binding moiety (TBM), a masking moiety (MM), anda cleavable moiety (CM). The ABP exhibits an “activatable” conformationsuch that the such that the TBM is less accessible to target whenuncleaved than after cleavage of the CM in the presence of a cleavingagent capable of cleaving the CM. The disclosure further provideslibraries of candidate ABPs, methods of screening to identify such ABPs,and methods of use. The disclosure further provides ABPs having a TBMthat binds VEGF, as well as compositions and methods of use.

Accordingly, the present disclosure provides an activatable bindingpolypeptide (ABP) comprising a target binding moiety (TBM); a maskingmoiety (MM) capable of inhibiting binding of the TBM to a target,wherein said MM does not have an amino acid sequence of a naturallyoccurring binding partner of said TBM; and a cleavable moiety (CM),wherein said CM is positioned in the activatable binding polypeptidesuch that in a cleaved state in the presence of a target, the TBM bindsthe target, and in an uncleaved state in the presence of the target,binding of the TBM to the target is inhibited by the MM.

In related embodiments, the MM is selected from a plurality of candidatepolypeptides based on its ability to inhibit binding of the TBM to thetarget in an uncleaved state and allow binding of the TBM to the targetin a cleaved state. In further related embodiments, the MM inhibitsbinding of the TBM to the target via steric hindrance when the ABP is inan uncleaved state. In other related embodiments, the MM comprises acysteine residue and steric hindrance is achieved via disulfide bondlinkage between said cysteine residue and an additional cysteine residueadjacent to or within the TBM. In additional embodiments the TBM is anextracellular polypeptide.

In further related embodiments, the CM is located between the TBM andthe MM in the ABP, and in other embodiments is located within the MM. Incertain embodiments, the CM comprises a protease substrate, which canbe, for example, a plasmin substrate, a caspase substrate or a matrixmetalloprotease (MMP) substrate (e.g., a substrate of MMP-1, MMP-2,MMP-9, or MMP-14). In other embodiments, the CM includes a proteasesubstrate is a substrate for an intracellular protease. In additionalembodiments, the CM comprises a cysteine-cysteine disulfide bond.

In another aspect, the disclosure provides methods of screening for anactivatable binding polypeptide (ABP), the method comprising contactinga plurality of candidate activatable binding polypeptides (candidateABPs) with a target capable of binding a target binding moiety of thecandidate ABPs and a cleaving agent capable of cleaving a cleavablemoiety (CM) of the ABPs; screening a first population of members of saidplurality which bind to said target in the presence of the cleavingagent; contacting said first population with the target in the absenceof the cleaving agent; and screening a second population of members fromsaid first population by depleting said first population for membersthat bind the target in the absence of the cleaving agent; wherein saidmethod provides for selection of candidate ABPs which exhibit decreasedbinding to the target in the absence of the cleaving agent as comparedto target binding in the presence of the cleaving agent.

In related embodiments the cleaving agent is a protease or a reducingagent. In further related embodiments, the target comprises a detectablelabel. In further embodiments, the first population is selected bydetection of the detectable label. In further embodiments, the secondpopulation is produced by separating from the first population membersthat are detectably labeled.

In further related embodiments, each of said plurality of candidateactivatable binding polypeptides is presented on a surface of areplicable biological entity in a display scaffold.

The disclosure further provides libraries of candidate activatablebinding polypeptides (ABPs), the library comprising a plurality ofcandidate ABPs displayed on the surface of a replicable biologicalentity. In related embodiments the replicable biological entity is abacterial, yeast or mammalian cell.

The disclosure also provides compositions comprising a nucleic acidconstruct comprising a nucleic acid coding for an ABP. In relatedembodiments, the nucleic acid construct further comprises a nucleic acidcoding for a display scaffold wherein the nucleic acid coding for theABP is operably inserted into the construct to provide for expression ofa fusion protein for presentation of the ABP in the display scaffold onthe surface of a host cell. An exemplary display scaffold is acircularly permuted outer membrane protein X (CPX). In relatedembodiments, the ABP is a candidate ABP having a candidate MM.

The disclosure further provides methods of making a library of candidateactivatable binding polypeptides, the method comprising introducing intogenomes of replicable biological entities a collection of recombinantDNA constructs that encode a plurality of candidate activatable bindingpolypeptides (ABPs), wherein each member of said plurality comprises atarget binding moiety (TBM), a cleavable moiety (CM) and a candidatemasking moiety (MM), said introducing producing recombinant replicablebiological entities; and culturing said recombinant replicablebiological entities under conditions suitable for expression and displayof the candidate ABPs.

The disclosure also provides pharmaceutical compositions comprising atherapeutically effective amount of an activatable binding polypeptide(ABP) and a pharmaceutically acceptable excipient. In relatedembodiments the TBM of the ABP is capable of binding VEGF to effect VEGFinhibition.

The disclosure also provides methods of inhibiting angiogenesis in asubject in need thereof, the method comprising administering to asubject in need thereof a therapeutically effective amount of an ABP,with exemplary ABPs including those having a TBM that binds VEGF toeffect inhibition of VEGF activity (e.g., at a tumor site).

In one embodiment, an ABP is disclosed wherein the target of the ABP isany one of VCAM-1, VEGF-A, CTLA-4 or CD40L.

The present disclosure also provides activatable binding polypeptides(ABPs), which contain a first target binding moiety (TBM), a second TBM,and a cleavable moiety (CM). The ABP exhibits an “activatable”conformation such that the such that at least one of the TBMs is lessaccessible to target when uncleaved than after cleavage of the CM in thepresence of a cleaving agent capable of cleaving the CM. The disclosurefurther provides libraries of candidate ABPs having such aconfiguration, methods of screening to identify such ABPs, and methodsof use. The disclosure further provides ABPs having a TBM that bindsVEGF and a TBM that binds FGF to effect inhibition of VEGF and FGFactivity, as well as compositions and methods of use.

Accordingly, the disclosure provides an activatable binding polypeptide(ABP) comprising a first target binding moiety (TBM); a second TBM; anda cleavable moiety (CM), wherein said CM is positioned in theactivatable binding polypeptide such that in a cleaved state in thepresence of a target, the first and second TBMs bind target, and in anuncleaved state the ABP is in a conformation such that the first TBMinterferes with target binding by the second TBM.

In related embodiments, the ABP in the uncleaved state is in aconformation such that the first and second TBMs interfere with bindingof target to the first and second TBMs. In further related embodiments,the first and second TBMs are capable of binding different targets,e.g., to FGF2 and to VEGF. In further related embodiments, the first TBMis selected from a plurality of candidate polypeptides based on theability of said first TBM to inhibit binding of said second TBM to atarget when the ABP is in an uncleaved state and allow binding of saidsecond TBM to the target when the ABP is in a cleaved state. In anotherembodiment, the first TBM interferes with target binding by said secondTBM via steric hindrance when the ABP is in an uncleaved state. Infurther related embodiments, the ABP comprises a first cysteine residuewithin or adjacent to said first TBM and a second cysteine residuewithin or adjacent to said second TBM, and wherein steric hindrance isachieved via disulfide bond linkage between said first and secondcysteine residue. In related embodiments, the target of said first TBMand said second TBM is an extracellular polypeptide. In otherembodiments, the CM is located between said first TBM and said secondTBM in the ABP, or, where the CM comprises a cysteine-cysteine pair, theCM can be located within either said first TBM or said second TBM. Infurther related embodiments, the CM comprises a protease substrate,e.g., a matrix metalloprotease (MMP) substrate, e.g., a substrate ofMMP-1, MMP-2, MMP-9, or MMP-14. In further related embodiments, theprotease substrate is a substrate for an intracellular protease. Inanother embodiment, the CM comprises a cysteine-cysteine disulfide bond.

The disclosure further provides methods for selecting for a dual targetbinding activatable binding polypeptide (ABP), said method comprising:contacting a plurality of candidate activatable binding polypeptides(ABPs), wherein each member of said plurality comprises a first targetbinding moiety (TBM), a second TBM and a cleavable moiety (CM), with atarget capable of binding said first TBM and a cleaving agent capable ofcleaving the CM; selecting a first population of members of saidplurality which bind to said target in the presence of the cleavingagent; contacting said first population with said target in the absenceof the cleaving agent; and selecting a second population of members fromsaid first population by depleting said first population for membersthat bind to said target in the absence of the cleaving agent; whereinsaid method provides for selection of candidate ABPs which exhibitdecreased binding to said target in the absence of the cleaving agent ascompared to binding to said target in the presence of the cleavingagent.

In related embodiments, the cleaving agent is a protease or a disulfidebond reducing agent. In further related embodiments, the first targetand the second target each comprise a detectable label. In furtherrelated embodiments, the first population is selected by detection ofthe detectable label. In further related embodiments, the secondpopulation is produced by separating from the first population membersthat are detectably labeled. In still other embodiments, the pluralityof candidate activatable binding polypeptides is presented on a surfaceof a replicable biological entity in a display scaffold. In furtherembodiments, binding of the second TBM to target is assessed byproviding the amino acid sequence of the second TBM in a displayscaffold and detecting binding of target to the second TBM.

The disclosure further provides libraries of candidate dual targetbinding activatable binding polypeptides (ABPs), said library comprisinga plurality of candidate dual target binding ABPs displayed on thesurface of a replicable biological entity. In related embodiments, thereplicable biological entity is a bacterial, yeast or mammalian cells.

The disclosure also provides compositions comprising a nucleic acidconstruct comprising a nucleic acid coding for a dual target bindingABP. In related embodiments, the nucleic acid construct furthercomprises a nucleic acid coding for a display scaffold wherein thenucleic acid coding for the ABP is operably inserted into the constructto provide for expression of a fusion protein for presentation of theABP in the display scaffold on the surface of a host cell. In furtherrelated embodiments, the display scaffold is a circularly permuted outermembrane protein X (CPX).

The disclosure also provides compositions comprising a nucleic acidconstruct comprising a nucleic acid encoding a candidate dual targetbinding activatable binding polypeptide, and further wherein saidcandidate activatable binding polypeptide comprises: (a) a first targetbinding moiety (TBM); (b) a cleavable moiety (CM); and (c) a second TBM,wherein the first TBM, CM and second TBM are positioned such that theability of said first TBM to inhibit binding of said second TBM to atarget in an uncleaved state and allow binding of said second TBM to thetarget in a cleaved state can be determined.

In related embodiments, the nucleic acid construct further comprises anucleic acid coding for a circularly permuted outer membrane protein X(CPX).

The disclosure further provides methods of making a library of candidatedual target binding activatable binding polypeptides, said methodcomprising introducing into genomes of replicable biological entities acollection of recombinant DNA constructs that encode a plurality of dualtarget binding candidate activatable binding polypeptides (ABPs),wherein each member of said plurality comprises a first target bindingmoiety (TBM), a cleavable moiety (CM) and a second TBM, said introducingproducing recombinant replicable biological entities; and culturing saidrecombinant replicable biological entities under conditions suitable forexpression and display of the candidate dual target binding ABPs.

The disclosure also provides pharmaceutical compositions comprising atherapeutically effective amount of a dual target binding activatablebinding polypeptide (ABP) and a pharmaceutically acceptable excipient.In related embodiments, the first TBM of the ABP binds VEGF to effectVEGF inhibition and the second TBM binds fibroblast growth factor-2(FGF2) to effect FGF2 inhibition.

The disclosure also provides methods of inhibiting angiogenesis in amammalian subject, method comprising administering to a subject in needthereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a dual target binding ABP.

In one aspect, the disclosure provides for a composition comprising anantigen binding domain (ABD) capable of binding a target, wherein saidABD is coupled to at least one masking moiety (MM) wherein said MMinterferes with specific binding of the ABD to the target. In oneembodiment the composition further comprises a cleavable moiety (CM)wherein said composition comprises two configurations, a firstconfiguration wherein the CM is in an uncleaved state and the MMinterferes with specific binding of the ABD to the target and a secondconfiguration wherein the CM is in a cleaved state and the MM does notinterfere with specific binding of the ABD to the target.

In another aspect the disclosure provides an activatable bindingpolypeptide (ABP) comprising at least one antigen binding domain (ABD)capable of binding a target, at least one masking moiety (MM) coupledsaid ABD capable of interfering with specific binding of the ABD to thetarget, and, at least one cleavable moiety (CM) coupled to said ABD,wherein said CM is positioned in the ABP such that in an uncleaved statethe MM interferes with specific binding of the ABD to the target and ina cleaved state the MM does not interfere with specific binding of theABD to the target. In certain embodiments the CM is coupled to theC-terminus of the ABD. In other embodiments, the CM is coupled to theN-terminus of the ABD or to the N-terminus of the VL or VH chains of theABD. In related embodiments the MM is coupled to the C-terminus of theABD. In other related embodiments, the MM is coupled to the N-terminusof the ABD or to the N-terminus of the VL or VH chains of the ABD. Infurther embodiments the ABP also contains a linker peptide positionedbetween the MM and the CM. In related embodiments a linker peptide ispositioned between the ABD and the CM. In specific embodiments the ABPcomprising an ABD, CM, and MM further comprises a detectable moiety. Incertain embodiments the detectable moiety is a diagnostic agent.

In one embodiment the ABP comprising an ABD, CM, and MM contains an ABDthat is from a full length antibody, Fab fragment, ScFv, or SCAB. Incertain embodiments the target of the ABD is selected from the groupconsisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3,EGFR, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B,IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R,ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30,CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR,TRAIL-R1, TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ,GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV,Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. Infurther embodiments the ABP comprising an ABD further comprises a CMwhich is a substrate for an enzyme selected from the group consisting ofMMP1, MMP2, MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, CATHEPSIN D,CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2,Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8,Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14,and TACE. In specific embodiments the CM is a substrate for MMP9.

In another aspect, the present disclosure provides a method of modifyinga composition containing an antigen binding domain (ABD) capable ofbinding a target, the method comprising coupling a masking moiety (MM)and a cleavable moiety (CM) to said ABD such that in a uncleaved statethe MM interferes with the ABD to specifically bind the target and in acleaved state the MM does not interfere with the ABD to specificallybind the target. In one embodiment the MM and/or the CM is coupled tothe C-terminus of the ABD. In another embodiment MM and/or the CMS iscoupled to the N-terminus of the ABD. In some embodiments the CM is asubstrate for an enzyme selected from the group consisting of MMP1,MMP2, MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, CATHEPSIN D,CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2,Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8,Caspase-9, Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14,and TACE.

In certain embodiments the target of the ABD is selected from the groupconsisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3,EGFR, FGF-2, FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B,IL1R, IL2, IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R,ERBB3, VCAM-1, CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30,CD33, CD40, CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR,TRAIL-R1, TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ,GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV,Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. Inrelated embodiments, the ABD is from a full length antibody, Fabfragment, ScFv, or SCAB and the ABD is from an antibody or an antibodyfragment to a target selected from the group consisting of VEGF, EGFR,CTLA-4, TNFα, Integrinα4, IL2R, Complement C5, CD11a, CD20, CD25, CD33,CD52, Glycoprotein receptor IIb/IIIa, IgE, Her2, and F protein of RSV.In further related embodiments ABD is from an antibody selected from thegroup consisting of bevacizumab, ranibizumab, trastuzumab, infliximab,adalimumab, efalizumab, gemtuzumab ozogamicin, tositumomab, ibritumomabtiuxetan, eculizumab, alemtuzumab, rituximab, abiciximab, cetuximab,daclizumab, basiliximab, gemtuzumab, panitumumab, eculizumab,natalizumab, omalizumab, ipilimumab, tremelimumab, and palivizumab. Inspecific embodiments the ABD is from an antibody or an antibody fragmentthereof to VEGF. In a related embodiment the ABD is from bevacizumab orranibizumab. In another specific embodiment, the ABD is from an antibodyor an antibody fragment thereof to TNFα. In a related embodiment, theABD is from infliximab or adalimumab. In another specific embodiment,the ABD is from an antibody or an antibody fragment thereof to CD20. Ina related embodiment the ABD is from tositumomab, ibritumomab tiuxetan,or rituximab. In yet another specific embodiment the ABD is from anantibody or an antibody fragment thereof to EGFR. In a relatedembodiment the ABD is from cetuximab or panitumumab. In yet anotherspecific embodiment the ABD is from an antibody or an antibody fragmentthereof to CTLA-4. In a related embodiment the ABD is from ipilimumab ortremelimumab.

The disclosure also provides a method of screening candidate peptides toidentify a masking moiety (MM) peptide with specific binding affinityfor an antibody or fragment thereof comprising an antigen binding domain(ABD). This method includes providing a library of peptide scaffolds,wherein each peptide scaffolds comprises a transmembrane protein (TM), acandidate peptide and involves contacting the antibody or fragmentthereof comprising an ABD with the library and identifying a MM peptidehaving specific binding affinity for the ABD contained in the antibodyor fragment thereof. In some embodiments the library comprises viruses,cells or spores. In a specific embodiment the library comprises E. coli.In certain embodiments the peptide scaffold further comprises adetectable moiety.

In certain embodiments the antibody or fragment thereof comprises an ABDto be screened in order to identify MMs is capable of binding a targetwherein the target is selected from the group consisting of VEGF-A,VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, EGFR, FGF-2, FGFR1,FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B, IL1R, IL2, IL2R,IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1,CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD40,CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1,TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ,GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV,Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. Inrelated embodiments the ABD to be screened to identify MMs is from anantibody selected from the group consisting of bevacizumab, ranibizumab,trastuzumab, infliximab, adalimumab, efalizumab, gemtuzumab ozogamicin,tositumomab, ibritumomab tiuxetan, eculizumab, alemtuzumab, rituximab,abiciximab, cetuximab, daclizumab, basiliximab, gemtuzumab, panitumumab,eculizumab, natalizumab, omalizumab, ipilimumab, tremelimumab, andpalivizumab. In specific embodiments the ABD is from an antibody or anantibody fragment to a target selected from the group consisting ofVEGF, EGFR, CTLA-4, TNFα, Integrinα4, IL2R, Complement C5, CD11a, CD20,CD25, CD33, CD52, Glycoprotein receptor IIb/IIIa, IgE, Her2, and Fprotein of RSV. In specific embodiments the ABD is from an antibody oran antibody fragment thereof to VEGF. In a related embodiment the ABD isfrom bevacizumab or ranibizumab. In another specific embodiment, the ABDis from an antibody or an antibody fragment thereof to TNFα. In arelated embodiment, the ABD is from infliximab or adalimumab. In anotherspecific embodiment, the ABD is from an antibody or an antibody fragmentthereof to CD20. In a related embodiment the ABD is from tositumomab,ibritumomab tiuxetan, or rituximab. In yet another specific embodimentthe ABD is from an antibody or an antibody fragment thereof to EGFR. Ina related embodiment the ABD is from cetuximab or panitumumab. In yetanother specific embodiment the ABD is from an antibody or an antibodyfragment thereof to CTLA-4. In a related embodiment the ABD is fromipilimumab or tremelimumab.

In other aspects the present disclosure provides methods of treatingand/or diagnosing a condition in a subject including administering tothe subject a composition comprising an antibody or fragment thereofcontaining an antigen binding domain (ABD) capable of binding a targetcoupled to a masking moiety (MM) and a cleavable moiety (CM), such thatin an uncleaved state the MM interferes with the ABD to specificallybind the target and in a cleaved state the MM does not interfere withthe ABD to specifically bind the target. In certain embodiments the ABDis from a full length antibody, a Fab fragment, ScFv, or SCAB. Incertain embodiments the ABD is from an antibody selected from the groupconsisting of bevacizumab, ranibizumab, trastuzumab, infliximab,adalimumab, efalizumab, gemtuzumab ozogamicin, tositumomab, ibritumomabtiuxetan, eculizumab, alemtuzumab, rituximab, abiciximab, cetuximab,daclizumab, basiliximab, gemtuzumab, panitumumab, eculizumab,natalizumab, omalizumab, ipilimumab, tremelimumab, and palivizumab. Inrelated embodiments the target is selected from the group consisting ofVEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFR1, VEGFR2, VEGFR3, EGFR, FGF-2,FGFR1, FGFR2, FGFR3, FGFR4, HER2/neu, DLL4, NOTCHR1, IL1B, IL1R, IL2,IL2R, IL4, IL6, IL12, IL13, IL15, IL18, IL23, IGF, IGF1R, ERBB3, VCAM-1,CXCR4, CD3, CD11a, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD40,CD40L, CD41, CD44, CD52, CD80, CD86, CTLA-4, TNFα, TNFR, TRAIL-R1,TRAIL-R2, IgE, IgE Receptor, PDGF-AA, PDGF-BB, PDGFRα, PDGFRβ,GPIIB/IIIA, CLAUDIN-3, CLAUDIN-4, C5 complement, F protein of RSV,Glyocprotein IIb/IIIa receptor, α4β1 integrin, and α4β7 integrin. Infurther related embodiments the ABD is from an antibody or an antibodyfragment to a target selected from the group consisting of VEGF, EGFR,CTLA-4, TNFα, Integrinα4, IL2R, Complement C5, CD11a, CD20, CD25, CD33,CD52, Glycoprotein receptor IIb/IIIa, IgE, Her2, and F protein of RSV.In specific embodiments the ABD is from an antibody or an antibodyfragment thereof to VEGF. In a related embodiment the ABD is frombevacizumab or ranibizumab. In another specific embodiment, the ABD isfrom an antibody or an antibody fragment thereof to TNFα. In a relatedembodiment, the ABD is from infliximab or adalimumab. In anotherspecific embodiment, the ABD is from an antibody or an antibody fragmentthereof to CD20. In a related embodiment the ABD is from tositumomab,ibritumomab tiuxetan, or rituximab. In yet another specific embodimentthe ABD is from an antibody or an antibody fragment thereof to EGFR. Ina related embodiment the ABD is from cetuximab or panitumumab. In yetanother specific embodiment the ABD is from an antibody or an antibodyfragment thereof to CTLA-4. In a related embodiment the ABD is fromipilimumab or tremelimumab.

In certain specific aspects the disclosure provides forenzyme-activatable antibodies or fragments thereof. In certainembodiments the enzyme selected from the group consisting of MMP1, MMP2,MMP3, MMP8, MMP9, MMP14, plasmin, PSA, PSMA, CATHEPSIN D, CATHEPSIN K,CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3,Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9,Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE. Inrelated embodiments the antibody fragment is from a full lengthantibody, or is a scFv, Fab, or SCAB. In one specific aspect thedisclosure provides for an enzyme-activatable anti-VEGF-A antibody orfragment thereof. In one specific embodiment the antibody isranibizumab. In another embodiment the antibody is activated by MMP9. Inanother aspect the disclosure provides for an enzyme-activatableanti-CTLA-4 antibody or fragment thereof. In one specific embodiment theantibody is ipilimumab or tremelimumab. In another embodiment theantibody is activated by MMP9. In yet another related aspect thedisclosure provides for an enzyme-activatable VCAM-1 antibody offragment thereof. In one specific embodiment the antibody is activatedby MMP9.

In one aspect, the disclosure provides a reaction mixture comprising anABP, a protease capable of cleaving said ABP, and a target of said ABP.

Other aspects and embodiments will be readily apparent upon reading thepresent disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an ABP which shows the ABP in an inactive(uncleaved state) and an active (cleaved state). An exemplary sequencefor the CM (PLGLAG (SEQ ID NO:9)) is presented.

FIG. 2 is a schematic of a library screening procedure which may be usedto identify and isolate ABPs.

FIG. 3 shows the amino acid sequence of a T7 control construct (SEQ IDNO:12) and shows that the VEGF binds the control construct in both thepresence and absence of enzyme. FIG. 3 also shows cleavage of an MMP-2substrate (CM) by MMP-2.

FIG. 4 shows the amino acid sequences of a cysteine constrained loop ABP(SEQ ID NO:13) and a GS control construct (SEQ ID NO:14). FIG. 4 alsoshows a diagram of the ABP in an inactive (uncleaved state) and anactive (cleaved state).

FIG. 5 shows the results of binding experiments which indicate that theformation of a cysteine constrained loop in an ABP interferes with VEGFbinding. Diagrams of the GS control and the ABP, in both cleaved anduncleaved states, are also shown.

FIG. 6 shows the amino acid sequences of 4 exemplary construct libraries(construct 1: SEQ ID NO:15; construct 2: SEQ ID NO:16; construct 3: SEQID NO:17; construct 4: SEQ ID NO:18) and shows diagrams representing thedisplayed constructs of the libraries.

FIG. 7 is a schematic of the screening procedure applied to theconstruct libraries shown in FIG. 6.

FIG. 8 shows that members of an exemplary library that exhibitswitch-like behavior can be identified after sorting constructsaccording to the screening procedure shown in FIG. 7.

FIG. 9 shows that selected library clones demonstrate improved switchingactivity over cysteine constrained controls.

FIG. 10 shows the amino acid sequences of clones isolated from thelibraries that demonstrated the most marked “switching” phenotype (clone2.2A.3: SEQ ID NO:107; 2.2A.4: SEQ ID NO:108; 2.2A.5: SEQ ID NO:109;2.2A.8: SEQ ID NO:110; 2.2A.19: SEQ ID NO:111; 1.2B.2: SEQ ID NO:112;4.2A.11: SEQ ID NO:113; 1.1: SEQ ID NO:114; 1.9: SEQ ID NO:115). Alsoshown is the amino acid sequence of the MM of each clone (clone 2.2A.3:SEQ ID NO:116; 2.2A.4: SEQ ID NO:117; 2.2A.5: SEQ ID NO:11; 2.2A.8: SEQID NO:118; 2.2A.19: SEQ ID NO:25; 1.2B.2: SEQ ID NO:119; 4.2A.11: SEQ IDNO:19; 1.1: SEQ ID NO:120; 1.9: SEQ ID NO:121).

FIG. 11 shows the results of binding experiments that demonstrateswitching activity for clones having cysteine residues in the MM as wellfor clones lacking cysteine residues in the MM.

FIG. 12 shows the results of experiments demonstrating that reduction ofdisulfide bonds in both a cysteine constrained parent and a libraryclone having a cysteine in the MM results in increased binding of VEGFto the TBM of the constructs.

FIG. 13 shows a graphical representation of the improvement in K_(d)that occurred when a cysteine constrained parent construct was contactedwith VEGF and treated with MMP-2, as compared with the K_(d) in theabsence of MMP-2 treatment.

FIG. 14 shows a graphical representation of the improvement in K_(d)that occurred when library clone 2.2A.5 was contacted with VEGF andtreated with MMP-2, as compared with the K_(d) in the absence of MMP-2treatment.

FIG. 15 shows the results of sorting of masking moiety Library 1 forexpanded dynamic range by adjusting the concentration of labeled target.Alternating A and B separations are performed with [VEGF] less than theKD of the VEGF in A sorts, and [VEGF]>KD in the B sorts.

FIG. 16 shows the results of sorting of masking moiety Library 1 forexpanded dynamic range by adjusting the concentration of labeled targetand identifies an average 4 fold dynamic range for a Library 1 ABP pool.The pool of EABPs resulting from sort 3A show a 4-fold average dynamicrange, indicating that some EABPs in the remaining pool will havegreater than 4× dynamic range. Note that for FIG. 16, the term EABPrefers to an ABP as described herein.

FIG. 17 shows a diagram of an exemplary embodiment of aprotease-activatable VEGF inhibitor.

FIG. 18 shows the amino acid sequence of an exemplary ABP identifiedthrough a screen of a candidate ABP library (SEQ ID NO:122).

FIG. 19 shows a diagram of candidate ABP libraries with candidatemasking moieties suitable for the identification of protease-activatableVEGF inhibitors.

FIG. 20 is a schematic showing exemplary ABPs of the present disclosure.

FIG. 21 shows an embodiment of the library screening procedure shown inFIG. 2.

FIG. 22 shows the sequences for various exemplary ABPs having cysteineand non-cysteine containing MMs (clone 2-2A-5: SEQ ID NO:123; 1-2B-2:SEQ ID NO:124; 1-3A-2: SEQ ID NO:125; 1-3A-3: SEQ ID NO:126; 1-3A-4: SEQID NO:127; 2-3B-5: SEQ ID NO:128; 2-3B-8: SEQ ID NO:129; 2-2A-8: SEQ IDNO:130; 2-2A-19: SEQ ID NO:131; 4-2A-11: SEQ ID NO:132; 2-3B-6: SEQ IDNO:133; 2-3B-7: SEQ ID NO:134).

FIG. 23 shows fluorescence values for a cysteine constrained loopstructure in the presence and absence of MMP-2 compared with thefluorescence values for library clones 4.2A.11 (a.k.a. 4-2A-11) and2.3B.5 (a.k.a. 2-3B-5) in the presence and absence of MMP-2.

FIG. 24 shows fold fluorescence increase after enzyme treatment forvarious ABP library clones.

FIG. 25 shows switching activity for selected ABP library clones.

FIG. 26 shows a diagram of a maltose-binding protein (MBP)-ABP fusionutilized in soluble protein binding assays.

FIG. 27 provides graphs showing Biacore™ assay results demonstratingthat soluble ABP fusions retain enzyme mediated binding properties.

FIG. 28 provides the results of FACS analysis showing binding ofselected candidate MM peptides to anti-VCAM-1 scFV.

FIGS. 29, 30 and 31 each provide an amino acid sequence of a propheticABP comprising an anti-VCAM-1 scFV (FIG. 29: SEQ ID NO:135; FIG. 30: SEQID NO:136; FIG. 31: SEQ ID NO:137).

FIGS. 32, 33 and 34 each provide an amino acid sequence of a propheticABP comprising an anti-VCAM-1 scFV, wherein the ABPs are designed forcytoplasmic expression as inclusion bodies (FIG. 32: SEQ ID NO:138; FIG.33: SEQ ID NO:139; FIG. 34: SEQ ID NO:140).

FIG. 35 provides a schematic showing activation and target binding of anABP with a TBM comprising an anti-VCAM-1 scFV ABD and a CM comprising anMMP-1 substrate.

FIG. 36 shows a protease-activated ABP containing an antigen bindingdomain (ABD).

FIG. 37 illustrates a process to produce a protease-activated ABPcontaining an antigen binding domain (ABD), involving: screening forMMs; screening for CMs; assembling the MM, CM, and TBM containing anABD; expressing and purifying the assembled construct; and assaying theassembled construct for activity and toxicity in vitro and in vivo.

FIG. 38 provides an exemplary MMP-9 cleavable masked anti-VEGF scFvamino acid sequence (SEQ ID NO:141).

FIG. 39 provides ELISA data showing the MMP-9 activation of theMBP:anti-VEGFscFv ABPs with the MMs 306 and 314. Samples were treatedwith TEV to remove the MBP fusion partner and subsequently activated byMMP-9 digestion.

FIG. 40 provides ELISA data demonstrating the MMP-9-dependent VEGFbinding of the anti-VEGFscFv His construct with the 306 MM.

FIG. 41 provides ELISA data demonstrating the MMP-9-dependent VEGFbinding of anti-VEGFscFv-Fc ABPs with the MMs 306 and 314 from HEK cellsupernatants.

FIG. 42 provides ELISA data showing the MMP-9-dependent VEGF binding ofanti-VEGF scFv-Fc ABP constructs with the MMs 306 and 314 that werepurified using a Protein A column.

FIG. 43 shows light and heavy chains of anti-CTLA4 joined via SOE-PCR togenerate scFv constructs in both orientiations, V_(H)V_(L) andV_(L)V_(H).

FIG. 44 illustrates the use of PCR to add sites for MM cloning, CMcleavage sequence, GGS2 linker on the N-terminus of the anti-CTLA4 scFvV_(H)V_(L) and V_(L)V_(H) constructs.

FIG. 45 provides the nucleotide sequence (SEQ ID NO:95) and amino acidsequence (SEQ ID NO:94) of a MM linker-CM-anti-CTLA4 scFv linker used inthe preparation of ABPs including an anti-CTLA4 scFv.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” “and,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “an activatable binding polypeptide” includes a pluralityof such activatable binding polypeptides and reference to “theactivatable binding polypeptide” includes reference to one or moreactivatable binding polypeptides and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The present disclosure provides activatable binding polypeptides (ABPs),which contain a target binding moiety (TBM), a masking moiety (MM), anda cleavable moiety (CM). The ABP exhibits an “activatable” conformationsuch that the TBM is less accessible to target when uncleaved than aftercleavage of the CM, e.g., in the presence of a cleavage agent (e.g., aprotease which recognizes the cleavage site of the CM). The disclosurefurther provides libraries of candidate ABPs, methods of screening toidentify such ABPs, and methods of use. The disclosure further providesABPs having a TBM that binds VEGF, as well as compositions and methodsof use.

The present disclosure also provides ABPs, which contain a first TBM, asecond TBM, and a CM. These ABPs exhibit an “activatable” conformationsuch that at least one of the TBMs is less accessible to target whenuncleaved than after cleavage of the CM in the presence of a cleavingagent capable of cleaving the CM. The disclosure further provideslibraries of candidate ABPs having such a configuration, methods ofscreening to identify such ABPs, and methods of use. The disclosurefurther provides ABPs having a TBM that binds VEGF and a TBM that bindsFGF to effect inhibition of VEGF and FGF activity, as well ascompositions and methods of use.

The present disclosure also provides activatable binding polypeptides(ABPs), which include a target binding moiety (TBM) that is an antibodyor an antibody fragment containing an antigen binding domain capable ofbinding a target (ABD), a masking moiety (MM), and a cleavable moiety(CM). The ABP exhibits an “activatable” conformation such that the ABDis less accessible to the target when uncleaved than after cleavage ofthe CM, e.g., in the presence of a cleavage agent (e.g., a proteasewhich recognizes the cleavage site of the CM). The disclosure furtherprovides libraries of candidate ABPs, candidate MMs for the ABD, methodsof screening to identify such ABPs and MMs and methods of use. Thedisclosure further provides ABPs having ABDs that bind one or more ofseveral targets disclosed herein as well as compositions and methods ofuse.

DEFINITIONS

The term “activatable binding polypeptide” or “ABP” generally refers toa polypeptide that contains a target binding moiety (TBM), a cleavablemoiety (CM), and a masking moiety (MM). The TBM generally contains anamino acid sequence that provides for binding to a target protein (e.g.,VEGF). In some embodiments the TBM comprises the antigen binding domain(ABD) of an antibody or antibody fragment thereof.

The CM generally includes an amino acid sequence that serves as thesubstrate for an enzyme and/or a cysteine-cysteine pair capable offorming a reducible disulfide bond. As such, when the terms “cleavage,”“cleavable,” “cleaved” and the like are used in connection with a CM,the terms encompass enzymatic cleavage, e.g., by a protease, as well asdisruption of a disulfide bond between a cysteine-cysteine pair viareduction of the disulfide bond that can result from exposure to areducing agent.

The MM is an amino acid sequence that, when the CM of the ABP is intact(i.e., uncleaved by a corresponding enzyme, and/or containing anunreduced cysteine-cysteine disulfide bond), the MM interferes withbinding of the TBM to its target. The amino acid sequence of the CM mayoverlap with or be included within the MM. It should be noted that forsake of convenience “ABP” is used herein to refer to an ABP in both itsuncleaved (or “native”) state, as well as in its cleaved state. It willbe apparent to the ordinarily skilled artisan that in some embodiments acleaved ABP may lack an MM due to cleavage of the CM, e.g, by aprotease, resulting in release of at least the MM (e.g., where the MM isnot joined to the ABP by a covalent bond (e.g., a disulfide bond betweencysteine residues). Exemplary ABPs are described in more detail below.

In an embodiment of particular interest, the ABP comprises two TBMs,wherein at least one of the TBMs acts as a masking moiety (MM) for theother TBM and/or the two TBMs serves as masking moieties for oneanother, such that in the uncleaved conformation, the ABP exhibitsreduced binding to a target for at least one of the TBMs relative towhen the ABP is in the cleaved conformation. Thus “activatable bindingpolypeptide” or “ABP” in this embodiment encompasses a polypeptide thatcontains a first target binding moiety (TBM), a second TBM, and acleavable moiety (CM), wherein the first and second TBMs interact to“mask” binding of at least one of the TBMs to target (i.e., the firstand/or second TBMs act as a masking moiety (MM) for target binding). TheTBM generally contains an amino acid sequence that provides for bindingto a target protein (e.g., VEGF).

In this latter embodiment, when the CM of the ABP is intact (i.e.,uncleaved by a corresponding enzyme, and/or containing an unreducedcysteine-cysteine disulfide bond), the interaction of the first andsecond TBMs interferes with binding of one or both of the TBMs to theircorresponding target(s). It should be noted that for sake of convenience“ABP” is used herein to refer to an ABP in both its uncleaved (or“native”) state, as well as in its cleaved state. It will be apparent tothe ordinarily skilled artisan that in some embodiments a cleaved ABPmay no longer contain two TBMs as described above due to cleavage of theCM, e.g, by a protease. Where the ABP includes both a protease-cleavableCM and a CM that includes a disulfide bond, cleavage of the proteasecleavable CM may leave the disulfide bond intact, and thus the ABP inthe cleaved form may retain two the TBMs, but in an “unmasked”configuration allowing for target binding. Exemplary ABPs are describedin more detail below.

As used herein, the term “cleaving agent” refers to an agent capable ofcleaving a sequence of the CM, e.g., by enzymatic cleavage, or areducing agent capable of reducing a disulfide bond between acysteine-cysteine pair. A “reducing agent” generally refers to acompound or element that serves as an electron-donating compound in areduction-oxidation reaction with a disulfide bond. Reducing agents ofparticular interest include cellular reducing agents such as proteins orother agents that are capable of reducing a disulfide bond underphysiological conditions, e.g., glutathione, thioredoxin, NADPH,flavins, and ascorbate.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include coded and non-coded amino acids, chemically or biochemicallymodified or derivatized amino acids, and polypeptides having modifiedpeptide backbones. The term includes fusion proteins, including, but notlimited to, fusion proteins with a heterologous amino acid sequence,fusions with heterologous and homologous leader sequences, with orwithout N-terminal methionine residues; immunologically tagged proteins;and the like.

The terms “protease”, “proteinase” and “enzyme capable of cleaving apolypeptide” are used interchangeably herein to refer to any enzyme,e.g., an endopeptidase or exopeptidase, usually an endopeptidase, thathydrolyzes peptide bonds.

The term “replicable biological entity” refers to self-replicatingbiological cells, including bacterial, yeast, protozoan, and mammaliancells, as well various viruses and bacteriophage capable of infectingsuch cells and replicating, and the like.

The terms “nucleic acid molecule” and “polynucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides, or analogsthereof. Polynucleotides may have any three-dimensional structure, andmay perform any function, known or unknown. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, exons, introns,messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence. Also encompassed are polypeptide sequences that areimmunologically identifiable with a polypeptide encoded by the sequence.

“Construct” is used herein to refer to a polypeptide or nucleic acidcharacterized as a covalently and operably linked elements. For example,an ABP construct can refer to a ABP polypeptide including at least aTBM, an MM, and a CM, which are operably linked to provide a switchablephenotype as described herein, as well as nucleic acid encoding such anABP polypeptide.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest in a host cell. Thus, the term includescloning, and expression vehicles, as well as integrating vectors.

As used herein, “recombinant” has the usual meaning in the art, andrefers to a polynucleotide synthesized or otherwise manipulated in vitro(e.g., “recombinant polynucleotide”), to methods of using recombinantpolynucleotides to produce gene products in cells or other biologicalsystems, or to a polypeptide (“recombinant protein”) encoded by arecombinant polynucleotide.

The term “recombinant” when used with reference to a cell indicates thatthe cell contains a heterologous nucleic acid, or expresses a peptide orprotein encoded by such a heterologous nucleic acid, and usuallyprovides for replication of such heterologous nucleic acid. Recombinantcells can contain genes that are not found within the native(non-recombinant) form of the cell. Recombinant cells can also containgenes found in the native form of the cell wherein the genes aremodified and re-introduced into the cell by artificial means. The termalso encompasses cells that contain a nucleic acid endogenous to thecell that has been modified without removing the nucleic acid from thecell; such modifications include those obtained by gene replacement,site-specific mutation, and related techniques.

A “heterologous sequence”, “heterologous nucleic acid”, “heterologouspolypeptide” or “heterologous amino acid sequence” as used herein, isone that originates from a source foreign to the particular host cell,or, if from the same source, is modified from its original form. Thus, aheterologous nucleic acid in a host cell includes nucleic acid that,although being endogenous to the particular host cell, has been modified(e.g., so that it encodes an amino acid sequence different from that ofa naturally-occurring or parent nucleic acid, to a nucleic acid toprovide a sequence not normally found in the host cell, and the like).Modification of the heterologous sequence can be accomplished by avariety of methods, e.g., by treating the DNA with a restriction enzymeto generate a DNA fragment that is capable of being operably linked tothe promoter or by operably linking the DNA to a heterologous promoterto provide an expression cassette that is not endogenous to the hostcell. Techniques such as site-directed mutagenesis are also useful formodifying a heterologous nucleic acid.

The term “operably linked” refers to functional linkage betweenmolecules to provide a desired function. For example, “operably linked”in the context of nucleic acids refers to a functional linkage betweennucleic acids to provide a desired function such as transcription,translation, and the like, e.g., a functional linkage between a nucleicacid expression control sequence (such as a promoter, signal sequence,or array of transcription factor binding sites) and a secondpolynucleotide, wherein the expression control sequence affectstranscription and/or translation of the second polynucleotide. “Operablylinked” in the context of a polypeptide refers to a functional linkagebetween amino acid sequences (e.g., of different domains) to provide fora described activity of the polypeptide (e.g., “masking” of a TBM whenan ABP is uncleaved; accessibility to protease to facilitate cleavageand “unmasking” of a TBM of an ABP; and the like).

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly and/orsynthetically, that has control elements that are capable of affectingexpression of a structural gene that is operably linked to the controlelements in hosts compatible with such sequences. Expression cassettesinclude at least promoters and optionally, transcription terminationsignals. Typically, the recombinant expression cassette includes atleast a nucleic acid to be transcribed and a promoter. Additionalfactors necessary or helpful in effecting expression can also be used asdescribed herein. For example, transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

“Introducing into a genome” as used herein in the context of making arecombinant replicable biological entity (e.g., host cell,bacteriophage) refers to production of a recombinant replicablebiological entity so as to provide for replication and, where desired,expression of an exogenous polypeptide with replication of thereplicable biological entity. Where the replicable biological entity isa host cell (e.g., a bacterial, yeast, or mammalian cell) “introducinginto a genome” encompasses both genomically introduction by genomicintegration (e.g., stable integration) as well as introduction of aepisomal element (e.g., plasmid) to provide for stable or transientmaintenance of the exogenous nucleic acid in the host cell. The term“transformation” is similarly used to indicate production of arecombinant cell by introduction of exogenous nucleic acid encoding apolypeptide of interest, where the exogenous nucleic acid can bemaintained stably or transiently as an episomal element (e.g., such as aplasmid in the context of a bacterial or yeast host cell) or can bestably or transiently genomically integrated.

The term “isolated” refers to separation of an entity (e.g.,polypeptide, nucleic acid, etc.) from other entities with which they arenaturally associated or may be associated during synthesis (e.g.,recombinant, chemical synthesis, etc.). The term “isolated” means anentity is not in a state in which it is found in nature or, where theentity is produced by recombinant or other synthetic means, is separatedor enriched relative to other components that may be present. Thus, forexample, an “isolated protein” is not as it appears in nature but may besubstantially less than 100% pure protein.

“Substantially pure” indicates that an entity (e.g., polypeptide) makesup greater than about 50% of the total content of the composition (e.g.,total protein of the composition) and typically, greater than about 60%of the total protein content. More typically, a “substantially pure”refers to compositions in which at least 75%, at least 75%, at least85%, at least 90% or more of the total composition is the entity ofinterest (e.g., of the total protein. Preferably, the protein will makeup greater than about 90%, and more preferably, greater than about 95%of the total protein in the composition.

“Enriched” indicates a composition is increased in the proportion of anentity of interest relative to a starting composition. For example, astarting or first population in which 10% of the members exhibit adesired attribute (e.g., enzymatically “switchable”) can be enriched toprovide a second population in which greater than 10% (e.g., 15% ormore) of the total members exhibit the desired activity. It should benoted that enrichment can result in a decrease of total differentmembers of the population such that enrichment is accompanied byselection.

“Screen” or “screening”, as well as the terms “selection” or“selecting”, are used herein to refer to treatment of a population so asto facilitate separation of members in the population having a desiredattribute (e.g., enzymatically ‘switchable”) from those that have a lessdesirable attribute (e.g., no detectable enzymatically switchablephenotype or an enzymatically switchable phenotype that is not of adesired dynamic range). A screen can be effected on a population ofmembers using one or more criterion. Screening can be accomplished bymeans that maintain the recoverability and/or viability of the separatedpopulations (e.g., by cell sorting using, e.g., FACS) or can beaccomplished by reducing viability or recoverability of undesiredmembers of the population.

A screen (or selection) can be a “positive screen” or a “negativescreen” (also referred to herein as a “positive selection” or a“negative selection”, respectively). In a “positive screen” membersexhibiting a desirable attribute are selected according to the presenceof a positive signal (e.g., the presence of a detectable signal, growthin the presence of an agent that inhibits growth of members deficient ina desirable attribute, etc.). In “negative screen” members exhibiting adesirable attribute are selected according to a decreased orundetectable signal (e.g., a relatively decreased or undetectablesignal; reduced growth in the presence of an agent that inhibits growthof members exhibiting a desirable attribute, etc.)

As used herein, “contacting” has its normal meaning and refers tocombining two or more entities (e.g., a target protein, a candidate ABP,an enzyme, etc.). Contacting can occur in, for example, a test tube orother container (e.g., combining of two or more agents [e.g., a cleavingagent (e.g., an enzyme) and a cell expressing a peptide displayscaffold]), in a cell-based system (e.g., contacting of a target proteinand/or cleaving agent (e.g., enzyme) with an ABP displayed on a cellsurface), or in a cell-free system (e.g., combining a cleaving agent(e.g., an enzyme) with a cell membranes, synthetic membrane, or othermembranes for presentation of a peptide display scaffold without theneed for intact cells).

As used herein, a “ligand” refers to a molecule(s) that binds to abinding partner molecule(s), e.g., a substrate, inhibitor, or allostericregulator binding to an enzyme, and includes natural and syntheticbiomolecules, such as proteins, polypeptides, peptides, nucleic acidmolecules, carbohydrates, sugars, lipids, lipoproteins, small molecules,natural and synthetic organic and inorganic materials, syntheticpolymers, and the like.

“Binding” as used herein generally refers to a covalent or non-covalentinteraction between two molecules (referred to herein as “bindingpartners”, e.g., a substrate and an enzyme), which binding is usuallyspecific.

As used herein, “specifically binds” or “binds specifically” refers tointeraction between binding partners such that the binding partners bindto one another, but do not bind other molecules that may be present inthe environment (e.g., in a biological sample, in tissue) at asignificant or substantial level under a given set of conditions (e.g.,physiological conditions).

As used herein, “fluorescent group” refers to a molecule that, whenexcited with light having a selected wavelength, emits light of adifferent wavelength. Fluorescent groups may also be referred to as“fluorophores”.

As used herein, the term “display scaffold” refers to a polypeptidewhich when expressed in a host cell is presented on an extracellularlyaccessible surface of the host cell and provides for presentation of anoperably linked heterologous polypeptide. For example, display scaffoldsfind use in the methods disclosed herein to facilitate screening ofcandidate ABPs. Display scaffolds can be provided such that aheterologous polypeptide of interest can be readily released from thedisplay scaffold, e.g. by action of a protease that facilitates cleavageof the fusion protein and release of a candidate ABP from the displayscaffold.

The term “detecting” or “assessing” includes any form of qualitative orquantitative measurement, and includes determining if an element ispresent or absent. The terms “determining”, “measuring”, “evaluating”,“assessing” and “assaying” are used interchangeably and includesquantitative and qualitative determinations. Assessing may be relativeor absolute. “Assessing the presence of” includes determining the amountof something present, and/or determining whether it is present orabsent. As used herein, the terms “detecting,” “determining,”“measuring,” and “assessing,” and “assaying” are used interchangeablyand include both quantitative and qualitative determinations.

The term “effective amount” or “therapeutically effective amount” meansa dosage sufficient to provide for treatment for the disease state beingtreated or to otherwise provide the desired effect (e.g., reduction intumor size, reduction in angiogenesis, etc.). The precise dosage willvary according to a variety of factors such as subject-dependentvariables (e.g., age, immune system health, etc.), the disease (e.g.,the type of cancer or tumor), and the treatment being effected.

The term “treatment site” is meant to refer to a site at which an ABP isdesigned to be switchable, as described herein, e.g., a site at which atarget for one or both TBMs of an ABP and a cleaving agent capable ofcleaving a CM of the ABP are co-localized. Treatment sites includetissues that can be accessed by local administration (e.g., injection,infusion (e.g., by catheter), etc.) or by systemic administration (e.g.,administration to a site remote from a treatment site). Treatment sitesinclude those that are relatively biologically confined (e.g., an organ,sac, tumor site, and the like).

Activatable Binding Polypeptides

The present disclosure provides activatable binding polypeptides (ABPs)which exhibit “activatable” binding, also referred to as “switchable”binding, to a target protein. ABPs generally include a target bindingmoiety (“TBM”), a masking moiety (“MM”) and a cleavable moiety (“CM”).In some embodiments, the CM contains an amino acid sequence that servesas a substrate for a protease of interest. In other embodiments, the CMprovides a cysteine-cysteine disulfide bond that is cleavable byreduction.

Schematics of ABPs are provided in FIGS. 1, 35 and 36, the latter twoschematically representing the embodiments where the TBM of the ABPcontains an antigen-binding domain (ABD). As illustrated in FIGS. 1, 35,and 36 the elements of the ABP are arranged such that the CM ispositioned such that in a cleaved state (or relatively “active” state)and in the presence of a target, the TBM binds a target, while in anuncleaved state (or relatively “inactive” state) in the presence of thetarget, binding of the TBM to the target is inhibited due to theconformation of the ABP, which can involve “masking” of the TBM by theMM. As used herein, the term “cleaved state” refers to the condition ofthe ABP following cleavage of the CM by a protease and/or reduction of acysteine-cysteine disulfide bond of the CM. The term “uncleaved state,”as used herein, refers to the condition of the ABP in the absence ofcleavage of the CM by a protease and/or in the absence reduction of acysteine-cysteine disulfide bond of the CM. As discussed above, “ABP” isused herein for sake of convenience to refer to ABP in both itsuncleaved (or “native”) state, as well as in its cleaved state. It willbe apparent to the ordinarily skilled artisan that in some embodiments acleaved ABP may lack an MM due to cleavage of the CM by protease,resulting in release of at least the MM (e.g., where the MM is notjoined to the ABP by a covalent bond (e.g., a disulfide bond betweencysteine residues).

By “activatable” or “switchable” is meant that the ABP exhibits a firstlevel of binding to a target when in a native or uncleaved state (i.e.,a first conformation), and a second level of binding to the target inthe cleaved state (i.e., a second conformation), where the second levelof target binding is greater than the first level of binding. Ingeneral, access of target to the TBM of the ABP is greater in thepresence of a cleaving agent capable of cleaving the CM than in theabsence of such a cleaving agent. Thus, in the native or uncleaved statethe TBM is “masked” from target binding (i.e., the first conformation issuch that it interferes with access of the target to the TBM), and inthe cleaved state the TBM is “unmasked” to target binding.

The CM and TBM of the ABP may be selected so that the TBM represents abinding moiety for a target of interest, and the CM represents asubstrate for a protease that is co-localized with the target at atreatment site in a subject. Alternatively or in addition, the CM is acysteine-cysteine disulfide bond that is cleavable as a result ofreduction of this disulfide bond. ABPs contain at least one of aprotease-cleavable CM or a cysteine-cysteine disulfide bond, and in someembodiments include both kinds of CMs. The ABPs disclosed herein findparticular use where, for example, a protease capable of cleaving a sitein the CM is present at relatively higher levels in target-containingtissue of a treatment site than in tissue of non-treatment sites.

Stated differently, the CM-TBM pair of the ABPs is designed to exploitthe elevated levels of a protease co-localized with a target. Thus, ABPcan be designed so that they are predominantly enzymatically activated(and thus exhibit higher levels of target binding) at a treatment sitethan at non-treatment sites in a subject. ABPs can thus provide forreduced toxicity and/or adverse side effects that can result frombinding of the TBM at non-treatment sites. Where the ABP contains a CMthat is cleavable by a reducing agent that facilitates reduction of adisulfide bond, the TBMs of such ABPs may selected to exploit activationof a TBM where a target of interest is present at a desired treatmentsite characterized by elevated levels of a reducing agent, such that theenvironment is of a higher reduction potential than, for example, anenvironment of a non-treatment site.

In general, an ABP can be designed by selecting a TBM of interest andconstructing the remainder of the ABP so that, when conformationallyconstrained, the MM provides for masking of the TBM. Structural designcriteria to be taken into account to provide for this functionalfeature. For example, where the ABP includes an MM, a at least one CM,and a linker, the linker is generally selected so that it is at least1.5 to 2 times, usually at least 2 times, the length representative ofthe distance from a target binding site of the TBM to a terminus of theTBM to which the remainder of the construct is to be attached.

Dual-target binding ABPs are of particular interest in the presentdisclosure. Such dual target binding ABPs contain two TBMs, which maybind the same or different target, and wherein at least one TBM servesas dual function of target binding and “masking”. As noted above, suchdual target binding ABPs generally contain two TBMs, wherein at leastone of the TBMs acts as a masking moiety (MM) for the other TBM and/orthe two TBMs serves as masking moieties for one another, such that inthe uncleaved conformation, the ABP exhibits reduced binding to a targetfor at least one of the TBMs relative to when the ABP is in the cleavedconformation. Thus “ABP” in this embodiment encompasses a polypeptidethat contains a first target binding moiety (TBM), a second TBM, and acleavable moiety (CM), wherein the first and second TBMs interact to“mask” binding of at least one of the TBMs to target (i.e., the firstand/or second TBMs act as a masking moiety (MM) for target binding). TheTBMs can include TBMs that bind to different targets (e.g., VEGF andfibroblast growth factor (FGF), e.g., FGF2). Each TBM can beindependently selected so that each contains 1 or more actives sites forbinding to the target.

FIG. 20 is a schematic showing an exemplary ABP of the presentdisclosure having two TBMs which can serve the dual function of bindingtheir respective targets when the ABP is cleaved and masking the ABPfrom binding to one or both targets of the TBMs when the ABP isuncleaved. In the upper portion of FIG. 20, an “MM” is shown, where theMM is a first TBM having a single binding site. In the center portion ofFIG. 20, an ABP having a first TBM and a second TBM (arbitrarily labeledas TBM1 and TBM2) with a cleavable moiety (CM) positioned between thetwo TBMs. In a switchable, uncleaved conformation (i.e., when the CM isintact such that it is uncleaved by a corresponding enzyme, and/orcontaining an unreduced cysteine-cysteine disulfide bond), asillustrated in the lower portion of FIG. 20, TBM1 interacts with TBM2thus “masking” binding to target to at least TBM1 and in particularembodiments masking target binding to both TBM1 and TBM2. When cleaved,each of the TBMs are “unmasked” and thus are free to bind theirrespective targets.

Dual target binding TBMs can be designed so as to have a CM cleavable bya cleaving agent that is co-localized in a target tissue with one orboth of the targets capable of binding to the TBMs of the ABP. Thedisclosure further contemplates multiple “units” of TBM1-TBM2 domains,such that cleavage of a single ABP by the cleaving agent results inrelease of multiple target binding fragments.

As exemplified in FIG. 20, a second CM can be positioned between the“masked” TBMs and a moiety of interest that can provide an additionaldesired function, such as targeting or serum half-life extension (asexemplified by a serum IgGbinding protein). This second CM can besusceptible to cleavage by the same or different cleaving agents.

It will be apparent to the ordinarily skilled artisan that in someembodiments a cleaved ABP may no longer contain two TBMs as describedabove due to cleavage of the CM, e.g, by a protease. Where the ABPincludes both a protease-cleavable CM and a CM that includes a disulfidebond, cleavage of the protease cleavable CM may leave the disulfide bondintact, and thus the ABP in the cleaved form may retain two the TBMs,but in an “unmasked” configuration allowing for target binding.Exemplary ABPs are described in more detail below.

ABPs exhibiting a switchable phenotype of a desired dynamic range fortarget binding in a cleaved versus uncleaved conformation are ofparticular interest. The term “dynamic range” as used herein generallyrefers to a ratio of (a) a maximum detected level of a parameter under afirst set of conditions to (b) a minimum detected value of thatparameter under a second set of conditions. For example, in the contextof an ABP, the dynamic range refers to the ratio of (a) a maximumdetected level of target protein binding to an ABP in the presence ofprotease capable of cleaving the CM of the ABP to (b) a minimum detectedlevel of target protein binding to an ABP in the absence of theprotease. The dynamic range of an ABP can be calculated as the ratio ofthe equilibrium dissociation constant of an ABP cleaving agent (e.g.,enzyme) treatment to the equilibrium dissociation constant of the ABPcleaving agent treatment. The greater the dynamic range of an ABP, thebetter the “switchable” phenotype of the ABP. ABPs having relativelyhigher dynamic range values (e.g., greater than 1) exhibit moredesirable switching phenotypes such that target protein binding by theABP occurs to a greater extent (e.g., predominantly occurs) in thepresence of a cleaving agent (e.g., enzyme) capable of cleaving the CMof the ABP than in the absence of a cleaving agent.

ABPs can be provided in a variety of structural configurations providedthat the TBM, MM and CM are operably positioned in the ABP such that aswitchable phenotype is provided. Exemplary formulae for ABPs areprovided below. It is specifically contemplated that the N- toC-terminal order of the TBM, MM and CM may be reversed within a ABP. Itis also specifically contemplated that the CM and MM may overlap inamino acid sequence, e.g., such that the CM is contained within the MM.

For example, ABPs can be represented by the following formula (In orderfrom an amino (“N”) terminal region to carboxyl (“C”) terminal region:(MM)-(CM)-(TBM)(TBM)-(CM)-(MM)

where MM is a masking moiety, CM is a cleavable moiety, and TBM is atarget binding moiety. It should be noted that although MM and CM areindicated as distinct components in the formula above, in all exemplaryembodiments (including formulae) disclosed herein it is contemplatedthat the amino acid sequences of the MM and the CM could overlap, e.g.,such that the CM is completely or partially contained within the MM. Inaddition, the formulae above provide for additional amino acid sequencesthat may be positioned N-terminal or C-terminal to the ABP elements. Insome embodiments, the dual target-binding ABP is such that the MM is asecond TBM. It is understood that throughout this disclosure, theformulae provided encompass such dual target-binding ABPs wherein “MM”in the formula is TBM1 and “TBM” is TBM2, where TBM1 and TBM2 arearbitrary designations for first and second TBMs, and where the targetcapable of binding the TBMs may be the same or different target, or thesame or different binding sites of the same target.

It is understood that generally ABPs can exhibit a switchable phenotypeas a result of folding of the ABP so that access of target to the TBM isinhibited by at least the MM. Thus, in many embodiments it may bedesirable to insert one or more linkers, e.g., flexible linkers, intothe ABP construct so as to provide for flexibility at one or more of theMM-CM junction, the CM-TBM junction, or both. For example, the TBM, MM,and/or CM may not contain a sufficient number of residues (e.g., Gly,Ser, Asp, Asn, especially Gly and Ser, particularly Gly) to provide thedesired flexibility. As such, the switchable phenotype of such ABPconstructs may benefit from introduction of one or more amino acids toprovide for a flexible linker. In addition, as described below, wherethe ABP is provided as a conformationally constrained construct, aflexible linker can be operably inserted to facilitate formation andmaintenance of a cyclic structure in the uncleaved ABP.

For example, in certain embodiments an ABP comprises one of thefollowing formulae (where the formula below represent an amino acidsequence in either N- to C-terminal direction or C- to N-terminaldirection):(MM)-L₁-(CM)-(TBM)(MM)-(CM)-L₁-(TBM)(MM)-L₁-(CM)-L₂-(TBM)cyclo[L₁-(MM)-L₂-(CM)-L₃-(TBM)]wherein MM, CM, and TBM are as defined above; wherein L₁, L₂, and L₃ areeach independently and optionally present or absent, are the same ordifferent flexible linkers that include at least 1 flexible amino acid(e.g., Gly); and wherein “cyclo” where present the ABP is in the form ofa cyclic structure due to the presence of a disulfide bond between apair of cysteines in the ABP. In addition, the formulae above providefor additional amino acid sequences that may be positioned N-terminal orC-terminal to the ABP elements. It should be understood that in theformula cyclo[L₁-(MM)-L₂-(CM)-L₃-(TBM)], the cysteines responsible forthe disulfide bond may be positioned in the ABP to allow for one or two“tails,” thereby generating a “lasso” or “omega” structure when the ABPis in a disulfide-bonded structure (and thus conformationallyconstrained state). The amino acid sequence of the tail(s) can providefor additional ABP features, such as binding to a target receptor tofacilitate localization of the ABP, increasing serum half-life of theABP, and the like. Targeting moieties (e.g., a ligand for a receptor ofa cell present in a target tissue) and serum half-life extendingmoieties (e.g., polypeptides that bind serum proteins, such asimmunoglobulin (e.g., IgG) or serum albumin (e.g., human serum albumin(HSA).

As noted above, the formula above encompass dual target-binding ABPssuch that “MM” in the formula is TBM1 and “TBM” is TBM2, where TBM1 andTBM2 are arbitrary designations for first and second TBMs, and where thetarget capable of binding the TBMs may be the same or different target,or the same or different binding sites of the same target. Thedisclosure further provides that the ABPs can include a second CM, suchthat the second CM is the same of different and provides for cleavablerelease of a moiety of interest (e.g., a targeting moiety, a serumhalf-life extending moiety, a moiety for immobilizing the ABP on asupport and the like).

Linkers suitable for use in ABPs are generally ones that provideflexibility of the ABP to facilitate a “masked” conformation. Suchlinkers are generally referred to as “flexible linkers”. Suitablelinkers can be readily selected and can be of any of a suitable ofdifferent lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS: SEQID NO: 1)_(n) and (GGGS: SEQ ID NO: 2)_(n), where n is an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers known in the art. Glycine and glycine-serine polymersare of interest since both of these amino acids are relativelyunstructured, and therefore may be able to serve as a neutral tetherbetween components. Glycine polymers are of particular interest sinceglycine accesses significantly more phi-psi space than even alanine, andis much less restricted than residues with longer side chains (seeScheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary flexiblelinkers include, but are not limited Gly-Gly-Ser-Gly: SEQ ID NO: 3,Gly-Gly-Ser-Gly-Gly: SEQ ID NO: 4, Gly-Ser-Gly-Ser-Gly: SEQ ID NO: 5,Gly-Ser-Gly-Gly-Gly: SEQ ID NO: 6, Gly-Gly-Gly-Ser-Gly: SEQ ID NO: 7,Gly-Ser-Ser-Ser-Gly: SEQ ID NO: 8, and the like. The ordinarily skilledartisan will recognize that design of an ABP can include linkers thatare all or partially flexible, such that the linker can include aflexible linker as well as one or more portions that confer lessflexible structure to provide for a desired ABP structure.

In addition to the element described above, the ABPs can containadditional elements such as, for example, amino acid sequence N- orC-terminal of the ABP. For example, ABPs can include a targeting moietyto facilitate delivery to a cell or tissue of interest. Moreover, in thecontext of the ABP libraries discussed further below, the ABP canprovided in the context of a scaffold protein to facilitate display ofthe ABP on a cell surface.

Exemplary basic elements of ABPs are described in more detail below.

Target Binding Moiety (TBM)

The target binding moiety (TBM) of ABPs can include any of a variety ofknown amino acid sequences that are capable of binding, usually capableof specifically binding, a target, usually a protein target, ofinterest. For example, the TBM can be selected to include the amino acidsequence of a binding partner of a target protein of interest, wherebinding of the binding partner and target provides for a desiredbiological effect, e g., inhibition of activity of the target proteinand/or detection of a target protein.

Exemplary classes of target proteins for which amino acid sequences ofbinding partners (e.g., inhibitors) are known include, but are notnecessarily limited to, cell surface receptors and secreted bindingproteins (e.g., growth factors), soluble enzymes, structural proteins(e.g. collagen, fibronectin) and the like. In specifically exemplaryembodiments, in no way limiting, the TBM is a binding partner for anytarget as listed in Table 1 below.

TABLE 1 Exemplary TBM targets VEGF-A HER2/neu IGF CD33 IgE ReceptorVEGF-B DLL4 IGF1R CD40 PDGF-AA VEGF-C NOTCHR1 ERBB3 CD40L PDGF-BB VEGF-DIL1B VCAM-1 CD44 PDGFRα VEGFR1 IL1R CXCR4 CD52 PDGFRβ VEGFR2 IL2 CD3CD80 GPIIB/IIIA VEGFR3 IL4 CD11a CD86 CLAUDIN-3 EGFR IL6 CD19 CTLA4CLAUDIN-4 FGF-2 IL12 CD20 TNFα C5 complement FGFR1 IL13 CD22 TNFR α4β1integrin FGFR2 IL15 CD25 TRAIL-R1 α4β7 integrin FGFR3 IL18 CD28 TRAIL-R2F protein of RSV FGFR4 IL23 CD30 IgE GP IIb/IIIa receptors IL2R CD41

In some embodiments, the TBM comprises a full length antibody or anantibody fragment containing an antigen binding domain, antigen bindingdomain fragment or an antigen binding fragment of the antibody (e.g., anantigen binding domain of a single chain) which is capable of binding,especially specific binding, to a target of interest, usually a proteintarget of interest. In this embodiment the TBM contains an antigenbinding domain (ABD). A schematic of an ABP containing a TBM thatcontains an ABD is provided in FIG. 36. In such embodiments, the ABD canbe binding polypeptides such as, but not limited to variable orhypervariable regions of light and/or heavy chains of an antibody (VL,VH), variable fragments (Fv), F(ab′) 2 fragments, Fab fragments, singlechain antibodies (scAb), single chain variable regions (scFv),complementarity determining regions (CDR), or other polypeptides knownin the art containing a ABD capable of binding target proteins orepitopes on target proteins. In further embodiments, the TBM may be achimera or hybrid combination containing a first TBM that contains a ABDand a second TBM that contains a ABD such that each ABD is capable ofbinding to the same or different target. In some embodiments, the TBM isa bispecific antibody or fragment thereof, designed to bind twodifferent antigens. In some embodiments there is a first MM and a secondMM coupled to the first TBM and the second TBM, respectively, in theactivatable form. The origin of the ABD can be a naturally occurringantibody or fragment thereof, a non-naturally occurring antibody orfragment thereof, a synthetic antibody or fragment thereof, a hybridantibody or fragment thereof, or an engineered antibody or fragmentthereof.

Methods for generating an antibody for a given target are well known inthe art. The structure of antibodies and fragments thereof, variableregions of heavy and light chains of an antibody (V_(H) and V_(L)), Fv,F(ab′)₂, Fab fragments, single chain antibodies (scAb), single chainvariable regions (scFv), and complementarity determining regions (CDR)are well understood. Methods for generating a polypeptide having adesired antigen-binding domain of a target antigen are known in the art.Methods for modifying antibodies to couple additional polypeptides arealso well-known in the art. For instance, peptides such as MMs, CMs orlinkers may be coupled to modify antibodies to generate the ABPs andother compositions of the disclosure. ABPs that containprotease-activated ABDs can be developed and produced with standardmethods, as described in the schematic in FIG. 37.

Exemplary classes of target proteins for which the TBM contains a ABDinclude, but are not necessarily limited to, cell surface receptors andsecreted binding proteins (e.g growth factors), soluble enzymes,structural proteins (e.g. collagen, fibronectin) and the like. Thetarget can be selected from any TBM target as described herein andexemplified but not limited to those in Table 1. In specific exemplaryembodiments, in no way limiting, exemplary sources for ABDs are listedin Table 2 below.

TABLE 2 Exemplary sources for ABDs Antibody Trade Name (antibody name)Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGFErbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™(infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab)Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab)Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20Zevalin ™ (ibritumomab tiuxetan) CD20 Rituxan ™ (rituximab) CD20Zenapax ™ (daclizumab) CD25 Myelotarg ™ (gemtuzumab) CD33 Mylotarg ™(gemtuzumab ozogamicin) CD33 Campath ™ (alemtuzumab) CD52 ReoPro ™(abiciximab) Glycoprotein receptor IIb/IIIa Xolair ™ (omalizumab) IgEHerceptin ™ (trastuzumab) Her2 Synagis ™ (palivizumab) F protein of RSV(ipilimumab) CTLA-4 (tremelimumab) CTLA-4

The exemplary sources for ABDs described in Table 2 are discussed ingreater detail in the following references which are incorporated byreference herein for their description of one or more of the referencedABD sources: Remicade™ (infliximab): U.S. Pat. No. 6,015,557, NagahiraK, Fukuda Y, Oyama Y, Kurihara T, Nasu T, Kawashima H, Noguchi C, OikawaS, Nakanishi T. Humanization of a mouse neutralizing monoclonal antibodyagainst tumor necrosis factor-alpha (TNF-alpha). J Immunol Methods. 1999Jan. 1; 222(1-2):83-92.) Knight D M, Trinh H, Le J, Siegel S, Shealy D,McDonough M, Scallon B, Moore M A, Vilcek J, Daddona P, et al.Construction and initial characterization of a mouse-human chimericanti-TNF antibody. Mol. Immunol. 1993 November; 30(16):1443-53. Humira™(adalimumab): Sequence in U.S. Pat. No. 6,258,562. Raptiva™(efalizumab): Sequence listed in Werther W A, Gonzalez T N, O'Connor SJ, McCabe S, Chan B, Hotaling T, Champe M, Fox J A, Jardieu P M, BermanP W, Presta L G. Humanization of an anti-lymphocyte function-associatedantigen (LFA)-1 monoclonal antibody and reengineering of the humanizedantibody for binding to rhesus LFA-1. J. Immunol. 1996 Dec. 1;157(11):4986-95. Mylotarg™ (gemtuzumab ozogamicin): (Sequence listed inCO MS, Avdalovic N M, Caron P C, Avdalovic M V, Scheinberg D A, Queen C:Chimeric and humanized antibodies with specificity for the CD33 antigen.J Immunol 148:1149, 1991) (Caron P C, Schwartz M A, Co M S, Queen C,Finn R D, Graham M C, Divgi C R, Larson S M, Scheinberg D A. Murine andhumanized constructs of monoclonal antibody M195 (anti-CD33) for thetherapy of acute myelogenous leukemia. Cancer. 1994 Feb. 1; 73(3Suppl):1049-56). Soliris™ (eculizumab): Hillmen P, Young N, Schubert J,Brodsky R, Socié G, Muus P, Röth A, Szer J, Elebute M, Nakamura R,Browne P, Risitano A, Hill A, Schrezenmeier H, Fu C, Maciejewski J,Rollins S, Mojcik C, Rother R, Luzzatto L (2006). “The complementinhibitor eculizumab in paroxysmal nocturnal hemoglobinuria”. N Engl JMed 355 (12): 1233-43. Tysabri™ (natalizumab): Sequence listed in LegerO J, Yednock T A, Tanner L, Horner H C, Hines D K, Keen S, Saldanha J,Jones S T, Fritz L C, Bendig M M. Humanization of a mouse antibodyagainst human alpha-4 integrin: a potential therapeutic for thetreatment of multiple sclerosis. Hum Antibodies. 1997; 8(1):3-16Synagis™ (palivizumab): Sequence listed in Johnson S, Oliver C, Prince GA, Hemming V G, Pfarr D S, Wang S C, Dormitzer M, O'Grady J, Koenig S,Tamura J K, Woods R, Bansal G, Couchenour D, Tsao E, Hall W C, Young JF. Development of a humanized monoclonal antibody (MEDI-493) with potentin vitro and in vivo activity against respiratory syncytial virus. JInfect Dis. 1997 November; 176(5):1215-24. Ipilimumab: J. Immunother:2007; 30(8): 825-830 Ipilimumab (Anti-CTLA4 Antibody) Causes Regressionof Metastatic Renal Cell Cancer Associated With Enteritis andHypophysitis; James C. Yang, Marybeth Hughes, Udai Kammula, RichardRoyal, Richard M. Sherry, Suzanne L. Topalian, Kimberly B. Suri,Catherine Levy, Tamika Allen, Sharon Mavroukakis, Israel Lowy, Donald E.White, and Steven A. Rosenberg Tremelimumab: Oncologist 2007; 12;873-883; Blocking Monoclonal Antibody in Clinical Development forPatients with Cancer; Antoni Ribas, Douglas C. Hanson, Dennis A. Noe,Robert Millham, Deborah J. Guyot, Steven H. Bernstein, Paul C. Canniff,Amarnath Sharma and Jesus Gomez-Navarro.

In some embodiments, the TBM of an ABP (including dual target-bindingABPs) comprises multiple active sites (e.g., 1, 2, 3, or more) forbinding a target. These active sites may have the same or differentamino acid sequences, and are usually designed to bind to differentbinding sites on a target of interest such that binding of a firstactive site of a TBM does not substantially interfere with binding of asecond active site of the TBM to the target. In certain embodiments theactive sites are separated by an amino acid linker sequence. A TBMcomprising multiple active sites is represented schematically in FIGS.17-19. ABPs can further include multiple TBM-MM “units”, which mayoptionally be separated by additional CMs so that upon exposure to acleaving agent, the one or more TBMs are unmasked. Dual target-bindingABPs can include multiple TBM1-TBM2 units, which units can be separatedby one or more CMs positioned on either “arm” of the dual target-bindingABP, and which may be cleavable by the same or different cleaving agent.

In certain embodiments, the TBM of an ABP can contain more than one ABD.In some embodiments the ABDs can be derived from bispecific antibodiesor fragments thereof. In other embodiments the ABP can be syntheticallyengineered so as to incorporate ABDs derived from two differentantibodies or fragments thereof. In such embodiments, the ABDs can bedesigned to bind two different targets, two different antigens, or twodifferent epitopes on the same target. A TBM containing multiple ABDscapable of binding more than one target site are usually designed tobind to different binding sites on a target or targets of interest suchthat binding of a first ABD of the TBM does not substantially interferewith binding of a second ABD of the TBM to a target. ABPs containingmultiple ABDs can further include multiple ABD-MM “units”, which mayoptionally be separated by additional CMs so that upon exposure to acleaving agent, the ABDs are unmasked. Dual target-binding ABPs caninclude multiple ABD1-ABD2 units, which units can be separated by one ormore CMs positioned on either “arm” of the dual target-binding ABP, andwhich may be cleavable by the same or different cleaving agent.

In general, ABPs contemplated by the present disclosure are those havinga TBM capable of binding an extracellular target, usually anextracellular protein target. However, ABPs can also be designed suchthat they are capable of cellular uptake and are designed to beswitchable inside a cell.

Masking Moiety (MM)

The masking moiety (MM) of an ABP generally refers to an amino acidsequence positioned in the ABP such that in an uncleaved state, even inthe presence of a target for the TBM, the MM interferes with binding ofthe TBM to the target. However, in the cleaved state of the ABP, theMM's interference with target binding to the TBM is reduced, therebyallowing greater access of the TBM to the target and providing fortarget binding. Thus, the MM is one that when the ABP is uncleavedprovides for “masking” of the TBM from target binding, but does notsubstantially or significantly interfere or compete for binding fortarget to the TBM when the ABP is in the cleaved conformation. Thus, thecombination of the MM and the CM facilitates the “switchable” phenotype,with the MM decreasing binding of target when the ABP is uncleaved, andcleavage of the CM by protease providing for increased binding oftarget.

The structural properties of the MM will vary according to a variety offactors such as the minimum amino acid sequence required forinterference with TBM binding to target, the target protein-TBM bindingpair of interest, the length of the TBM, the length of the CM, whetherthe CM is positioned within the MM and also serves to “mask” the TBM inthe uncleaved ABP, the presence or absence of linkers, the presence orabsence of a cysteine within or flanking the TBM that is suitable forproviding a CM of a cysteine-cysteine disulfide bond, and the like.

In some embodiments, the MM is coupled to the ABP by covalent binding.In one such embodiment, the coupling is to a C-terminus of the ABP. Inanother embodiment, the coupling is by cross-linking to an internalamino acid of the ABP. In another embodiment, the ABP composition ismasked by binding the MM to an N-terminus of the ABP. In yet anotherembodiment, the ABP is coupled to the MM by cysteine-cysteine disulfidebridges between the MM and the ABP.

The MM can be provided in a variety of different forms. For example, theMM can be selected to be a known binding partner of the TBM, providedthat the MM binds the TBM with less affinity and/or avidity than thetarget protein to which the TBM is designed to bind following cleavageof the CM so as to reduce interference of MM in target-TBM binding.Stated differently, as discussed above, the MM is one that “masks” theTBM from target binding when the ABP is uncleaved, but does notsubstantially or significantly interfere or compete for binding fortarget when the ABP is in the cleaved conformation. In a specificembodiment, the TBM and MM do not contain the amino acid sequences of anaturally-occurring binding partner pair, such that at least one of theTBM and MM does not have the amino acid sequence of a member of anaturally occurring binding partner. In a specific embodiment, the TBMand MM are other than a binding partner pair of TNF-alpha and a completeor partial extracellular domain of TNF receptor, or derivatives thereof,that act as a binding partner for TNF-alpha. In another specificembodiment, the TBM and MM are other than a binding partner pair ofTNF-alpha and the viral T2 protein, or derivatives thereof, that act asa binding partner for TNF-alpha. In another specific embodiment, the TBMand MM are other than a binding partner pair of FasL and a complete orpartial extracellular domain of a Fas receptor or derivatives thereof,that act as a binding partner for FasL. In another specific embodiment,the TBM and MM are other than a binding partner pair of FasL and a viralprotein or derivatives thereof, that act as a binding partner for FasL.In another specific embodiment, the TBM and MM are other than a bindingpartner pair of FasL and an antibody or fragment thereof having bindingaffinity for FasL.

For example, the TBM and MM can also be selected so they are not naturalbinding partners, where the MM may be, for example, a modified bindingpartner for the TBM which contains amino acid changes that at leastslightly decrease affinity and/or avidity of binding to the TBM suchthat, following cleavage, the MM does not substantially or significantlyinterfere with TBM-target binding. Because ABPs can be based on knownbinding partners for which the amino acid sequences that facilitatebinding are known, production of such MM-TBM pairs is well within theskill of the ordinarily skilled artisan. For example, the amino acidsequences that facilitate interaction of VEGF and a VEGF inhibitor arewell known, and are exemplified herein.

The MM can be identified through a screening procedure from a library ofcandidates ABPs having variable MMs. For example, a TBM and CM can beselected to provide for a desired enzyme/target combination, and theamino acid sequence of the MM can beidentified by the screeningprocedure described below to identify an MM that provides for aswitchable phenotype. For example, a random peptide library (e.g., fromabout 4 to about 40 amino acids or more) may be used in the screeningmethods disclosed herein to identify a suitable MM. A random peptidelibrary may also be utilized in connection with the targetedintroduction of cysteine residues to favor disulfide bond formation andfacilitate formation of a conformationally constrained, “cyclic” ABPstructure.

In other embodiments, MMs with specific binding affinity for an antigenbinding domain (ABD) can be identified through a screening procedurethat includes providing a library of peptide scaffolds consisting ofcandidate MMs wherein each scaffold is made up of a transmembraneprotein and the candidate MM. The library is then contacted with anentire or portion of TBM such as a full length antibody, a naturallyoccurring antibody fragment, or a non-naturally occurring fragmentcontaining an antigen binding domain (also capable of binding the targetof interest), and identifying one or more candidate MMs havingdetectably bound ABD. Screening can include one more rounds ofmagnetic-activated sorting (MACS) or fluorescence-activated sorting(FACS).

In this manner, ABPs having an MM that inhibits binding of the TBM tothe target in an uncleaved state and allows binding of the TBM to thetarget in a cleaved state can be identified, and can further provide forselection of an ABP having an optimal dynamic range for the switchablephenotype. Methods for identifying ABPs having a desirable switchingphenotype are described in more detail below.

Alternatively, the MM may not specifically bind the TBM, but ratherinterfere with TBM-target binding through non-specific interactions suchas steric hindrance. For example, the MM may be positioned in theuncleaved ABP such that the folded ABP allows the MM to “mask” the TBMthrough charge-based interaction, thereby holding the MM in place tointerfere with target access to the TBM.

ABPs can also be provided in a conformationally constrained structure,such as a cyclic structure, to facilitate the switchable phenotype. Thiscan be accomplished by including a pair of cysteines in the ABPconstruct so that formation of a disulfide bond between the cysteinepairs places the ABP in a loop or cyclic structure. Thus the ABP remainscleavable by the desired protease while providing for inhibition oftarget binding to the TBM. Upon cleavage of the CM, the cyclic structureis “opened”, allowing access of target to the TBM. FIG. 6 provides aschematic of an uncleaved ABP which is conformationally constrained by adisulfide bond (represented by a dashed line) between cysteine residuespositioned in a region at or near the ends of a ABP (where the “ends”refers to the ABP in the linear form prior to disulfide bond formation).Such cyclic ABPs can be designed or be optimized (e.g., using thescreening methods described below) such that in the uncleaved ABPaccessibility of the CM to its corresponding protease is greater thanaccessibility of the TBM to target protein binding. It should be notedthat target access to the TBM may also occur following reduction of thedisulfide bond.

The cysteine pairs can be positioned in the ABP at any position thatprovides for a conformationally constrained ABP, but that, following CMcleavage, does not substantially or significantly interfere with targetbiding to the TBM. For example, the cysteine residues of the cysteinepair are positioned in the MM and a linker flanked by the MM and TBM,within a linker flanked by the MM and TBM, or other suitableconfigurations. For example, the MM or a linker flanking an MM caninclude one or more cysteine residues, which cysteine residue forms adisulfide bridge with a cysteine residue positioned opposite the MM whenthe ABP is in a folded state. It is generally desirable that thecysteine residues of the cysteine pair be positioned outside the TBM soas to avoid interference with target binding following cleavage of theABP. Where a cysteine of the cysteine pair to be disulfide bonded ispositioned within the TBM, it is desirable that it be positioned to asto avoid interference with TBM-target binding following exposure to acleaving agent, e.g., after exposure to a reducing agent.

Exemplary ABPs capable of forming a cyclic structure by disulfide bondsbetween cysteines can be of the general formula (which may be fromeither N- to C-terminal or from C- to N-terminal direction):X_(n1)-(Cys₁)-X_(m)-CM-TBM-(Cys₂)-X_(n2)X_(n1)-cyclo[(Cys₁)-X_(m)-CM-TBM-(Cys₂)]-X_(n2)wherein

X_(n1) and X_(n2) are independently, optionally present or absent and,when present, independently represent any amino acid, and can optionallyinclude an amino acid sequence of a flexible linker (e.g., at least oneGly, Ser, Asn, Asp, usually at least one Gly or Ser, usually at leastone Gly), and n₁ and n₂ are independently selected from s zero or anyinteger, usually nor more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Cys₁ and Cys₂ represent first and second cysteines of a pair capable offorming a disulfide bond;

X_(m) represents amino acids of a masking motif (MM), where X is anyamino acid, wherein X_(m) can optionally include a flexible linker(e.g., at least one Gly, Ser, Asn, Asp, usually at least one Gly or Ser,usually at least one Gly); and where m is an integer greater than 1,usually 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (as described above);

CM represents a cleavable moiety (as described herein); and

TBM represents a target binding moiety (as described herein).

As used in the formula above, “cyclo” indicates a disulfide bond in theABP that provides for a cyclic structure of the ABP. Furthermore, theformula above contemplate dual target-binding ABPs wherein “MM” refersto a TBM1 and “TBM” refers to TBM2, where TBM1 and TBM2 are arbitrarydesignations for first and second TBMs, and where the target capable ofbinding the TBMs may be the same or different target, or the same ordifferent binding sites of the same target. In such embodiments, theTBM1 and/or TBM2 acts as a masking moiety to interfere with targetbinding to an uncleaved dual target-binding ABP.

As illustrated above, the cysteines can thus be positioned in the ABPallow for one or two “tails” (represented by X_(n1) and X_(n2) above),thereby generating a “lasso” or “omega” structure when the ABP is in adisulfide-bonded structure (and thus conformationally constrainedstate). The amino acid sequence of the tail(s) can provide foradditional ABP features, such as binding to a target receptor tofacilitate localization of the ABP.

For example, in an ABP containing an exemplary VEGF binder as a TBM, theTBM can comprise the amino acid sequence NFGYGKWEWDYGKWLEKVGGC: SEQ IDNO: 10, and a corresponding MM comprises the amino acid sequence PEWGCG:SEQ ID NO: 11. Further specific examples are provided in the Examplessection below.

In certain specific embodiments, the MM does not inhibit cellular entryof the ABP.

Cleavable Moiety (CM)

The cleavable moiety (CM) of the ABP may include an amino acid sequencethat can serve as a substrate for a protease, usually an extracellularprotease (i.e., other than an intracellular protease). Optionally, theCM comprises a cysteine-cysteine pair capable of forming a disulfidebond, which can be cleaved by action of a reducing agent. The CM ispositioned in the ABP such that when the CM is cleaved by a cleavingagent (e.g., a protease substrate of a CM is cleaved by the proteaseand/or the cysteine-cysteine disulfide bond is disrupted via reductionby exposure to a reducing agent), in the presence of a target, resultingin a cleaved state, the TBM binds the target, and in an uncleaved state,in the presence of the target, binding of the TBM to the target isinhibited by the MM. It should be noted that the amino acid sequence ofthe CM may overlap with or be included within the MM, such that all or aportion of the CM facilitates “masking” of the TBM when the ABP is inthe uncleaved conformation.

As discussed above, the CM may be selected based on a protease that isco-localized in tissue with the desired target of the TBM of the ABP. Avariety of different conditions are known in which a target of interestis co-localized with a protease, where the substrate of the protease isknown in the art. For example, the target tissue can be a canceroustissue, particularly cancerous tissue of a solid tumor. There are manyreports in the literature of increased levels of proteases having knownsubstrates in a number of cancers, e.g., solid tumors. See, e.g., LaRocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Furthermore,anti-angiogenic targets, such as VEGF, are known. As such, where the TBMof an ABP is selected such that it is capable of binding ananti-angiogenic target such as VEGF, a suitable CM will be one whichcomprises a peptide substrate that is cleavable by a protease that ispresent at the cancerous treatment site, particularly that is present atelevated levels at the cancerous treatment site as compared tonon-cancerous tissues. For example, the TBM of an ABP can be apolypeptide, peptide, or antigen binding domain (ABD) that binds VEGFand the CM can be a matrix metalloprotease (MMP) substrate, and thus iscleavable by an MMP.

Exemplary substrates can include but are not limited to substratescleavable by one or more of the following enzymes: MMP-1, MMP-2, MMP-3,MMP-8, MMP-9, MMP-14, PLASMIN, PSA, PSMA, CATHEPSIN D, CATHEPSIN K,CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3,Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9,Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.

Alternatively or in addition, the TBM of an ABP can be one that bindsVEGF and the CM can involve a disulfide bond of a cysteine pair, whichis thus cleavable by a reducing agent such as, for example, a cellularreducing agent such as glutathione (GSH), thioredoxins, NADPH, flavins,ascorbate, and the like, which can be present in large amounts in tissueof or surrounding a solid tumor.

Exemplary ABPs

In certain embodiments the ABP is an activatable antibody or activatableantibody fragment that includes a TBM, a CM, and a MM. In suchembodiments the TBM comprises an ABD or ABD fragment. Non limitingexemplary activatable antibody compositions include a MMP-9 activatable,masked anti-VEGF scFv, a MMP-9 activatable, masked anti-VCAM scFv, and aMMP-9 activatable masked anti-CTLA4. These are provided by way ofexample only and such enzyme activatable masked antibody ABPs could bedesigned to any target as listed in but not limited to those in Table 1and by using any antibody as listed in but not limited to those in Table2.

Methods and Compositions for Identifying and/or Optimizing ABPs

Methods for identifying and/or optimizing ABPs, as well as compositionsuseful in such methods, are described below.

Libraries of ABPs or Candidate ABPs Displayed on Replicable BiologicalEntities

In general, the screening methods to identify an ABP and/or to optimizean ABP for a switchable phenotype involve production of a library ofreplicable biological entities (as exemplified by cells) that display ontheir surface a plurality of different candidate ABPs. These librariescan then be subjected to screening methods to identify candidate ABPshaving one or more desired characteristics of an ABP.

The candidate ABP libraries can contain candidate ABPs that differ byone or more of the MM, linker (which may be part of the MM), CM (whichmay be part of the MM), and TBM. As discussed above, ABPs are may bedesigned to target a known protease-target pair of a condition ofinterest. Thus, generally the candidate ABPs in the library are variablefor the MM and/or the linker, with the TBM and CM being preselected.Where the ABP is to include pairs of cysteine residues to provide adisulfide bond in the ABP, the relative position of the cysteines in theABP can be varied.

The library for screening is generally provided as a library ofreplicable biological entities which display on their surface differentcandidate ABPs. For example, a library of candidate ABPs can include aplurality of candidate ABPs displayed on the surface of population of areplicable biological entities, wherein each member of said plurality ofcandidate activatable binding polypeptides comprises: (a) a targetbinding moiety (TBM); (b) a cleavable moiety (CM); and (c) a candidatemasking moiety (candidate MM), wherein the TBM, CM and candidate MM arepositioned such that the ability of the candidate MM to inhibit bindingof the TBM to a target in an uncleaved state and allow binding of theTBM to the target in a cleaved state can be determined.

Suitable replicable biological entities include cells (e.g., bacteria(e.g., E. coli), yeast (e.g., S. cerevesiae), mammalian cells),bacteriophage, and viruses. Bacterial host cells and bacteriophage,particularly bacterial host cells, are of interest.

Display of Candidate ABPs on the Surface of Replicable BiologicalEntities

A variety of display technologies using replicable biological entitiesare known in the art. These methods and entities include, but are notlimited to, display methodologies such as mRNA and ribosome display,eukaryotic virus display, and bacterial, yeast, and mammalian cellsurface display. See Wilson, D. S., et al. 2001 PNAS USA98(7):3750-3755; Muller, O. J., et al. (2003) Nat. Biotechnol. 3:312;Bupp, K. and M. J. Roth (2002) Mol. Ther. 5(3):329 3513; Georgiou, G.,et al., (1997) Nat. Biotechnol. 15(1):29 3414; and Boder, E. T. and K.D. Wittrup (1997) Nature Biotech. 15(6):553 557. Surface display methodsare attractive since they enable application of fluorescence-activatedcell sorting (FACS) for library analysis and screening. See Daugherty,P. S., et al. (2000) J. Immuunol. Methods 243(1 2):211 2716; Georgiou,G. (2000) Adv. Protein Chem. 55:293 315; Daugherty, P. S., et al. (2000)PNAS USA 97(5):2029 3418; Olsen, M. J., et al. (2003) Methods Mol. Biol.230:329 342; Boder, E. T. et al. (2000) PNAS USA 97(20):10701 10705;Mattheakis, L. C., et al. (1994) PNAS USA 91(19): 9022 9026; and Shusta,E. V., et al. (1999) Curr. Opin. Biotech. 10(2):117 122. Additionaldisplay methodologies which may be used to identify a peptide capable ofbinding to a biological target of interest are described in U.S. Pat.No. 7,256,038, the disclosure of which is incorporated herein byreference.

Phage display involves the localization of peptides as terminal fusionsto the coat proteins, e.g., pIII, pIIV of bacteriophage particles. SeeScott, J. K. and G. P. Smith (1990) Science 249(4967):386 390; andLowman, H. B., et al. (1991) Biochem. 30(45):10832 10838. Generally,polypeptides with a specific function of binding are isolated byincubating with a target, washing away non-binding phage, eluting thebound phage, and then re-amplifying the phage population by infecting afresh culture of bacteria.

Exemplary phage display and cell display compositions and methods aredescribed in U.S. Pat. Nos. 5,223,409; 5,403,484; 7,118,879; 6,979,538;7,208,293; 5571698; and 5,837,500.

Additional exemplary display scaffolds and methods include thosedescribed in U.S. Patent Application Publication No: 2007/0065878,published Mar. 22, 2007.

Optionally, the display scaffold can include a protease cleavage site(different from the protease cleavage site of the CM) to allow forcleavage of an ABP or candidate ABP from a surface of a host cell.

In one, where the replicable biological entity is a bacterial cell,suitable display scaffolds include circularly permuted Esccherichia coliouter membrane protein OmpX (CPX) described by Rice et al, Protein Sci.(2006) 15: 825-836. See also, U.S. Pat. No. 7,256,038, issued Aug. 14,2007.

Constructs Encoding ABPs and Candidate ABPs

The disclosure further provides nucleic acid constructs which includesequences coding for ABPs and/or candidate ABPs. Suitable nucleic acidconstructs include, but are not limited to, constructs which are capableof expression in a prokaryotic or eukaryotic cell. Expression constructsare generally selected so as to be compatible with the host cell inwhich they are to be used.

For example, non-viral and/or viral constructs vectors may be preparedand used, including plasmids, which provide for replication of ABP- orcandidate ABP-encoding DNA and/or expression in a host cell. The choiceof vector will depend on the type of cell in which propagation isdesired and the purpose of propagation. Certain constructs are usefulfor amplifying and making large amounts of the desired DNA sequence.Other vectors are suitable for expression in cells in culture. Thechoice of appropriate vector is well within the skill of the art. Manysuch vectors are available commercially. Methods for generatingconstructs can be accomplished using methods well known in the art.

In order to effect expression in a host cell, the polynucleotideencoding an ABP or candidate ABP is operably linked to a regulatorysequence as appropriate to facilitate the desired expression properties.These regulatory sequences can include promoters, enhancers,terminators, operators, repressors, and inducers. Expression constructsgenerally also provide a transcriptional and translational initiationregion as may be needed or desired, which may be inducible orconstitutive, where the coding region is operably linked under thetranscriptional control of the transcriptional initiation region, and atranscriptional and translational termination region. These controlregions may be native to the species from which the nucleic acid isobtained, or may be derived from exogenous sources.

Promoters may be either constitutive or regulatable. In some situationsit may be desirable to use conditionally active promoters, such asinducible promoters, e.g., temperature-sensitive promoters. Inducibleelements are DNA sequence elements that act in conjunction withpromoters and may bind either repressors (e.g. lacO/LAC Iq repressorsystem in E. coli) or inducers (e.g. gal1/GAL4 inducer system in yeast).In such cases, transcription is virtually “shut off” until the promoteris derepressed or induced, at which point transcription is “turned-on.”

Constructs, including expression constructs, can also include aselectable marker operative in the host to facilitate, for example,growth of host cells containing the construt of interest. Suchselectable marker genes can provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture.

Expression constructs can include convenient restriction sites toprovide for the insertion and removal of nucleic acid sequences encodingthe ABP and/or candidate ABP. Alternatively or in addition, theexpression constructs can include flanking sequences that can serve asthe basis for primers to facilitate nucleic acid amplification (e.g.,PCR-based amplification) of an ABP-coding sequence of interest.

The above described expression systems may be employed with prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. In some embodiments, a unicellular organism,such as E. coli, B. subtilis, S. cerevisiae, insect cells in combinationwith baculovirus vectors, or cells of a higher organism such asvertebrates, e.g. COS 7 cells, HEK 293, CHO, Xenopus Oocytes, etc., maybe used as the expression host cells. Expression systems for each ofthese classes and types of host cells are known in the art.

Methods of Making Libraries of ABPs or Candidate ABPs Displayed onReplicable Biological Entities

The present disclosure contemplates methods of making the libraries ofABPs and/or candidate ABPs described herein.

In one embodiment, a method of making an ABP library and/or candidateABP library comprises: (a) constructing a set of recombinant DNA vectorsas described herein that encode a plurality of ABPs and/or candidateABPs; (b) transforming host cells with the vectors of step (a); and (c)culturing the host cells transformed in step (b) under conditionssuitable for expression and display of the fusion polypeptides.

Production of Nucleic Acid Sequences Encoding Candidate ABPs

Production of candidate ABPs for use in the screening methods can beaccomplished using methods known in the art. Polypeptide display, singlechain antibody display, antibody display and antibody fragment displayare methods well know in the art. In general, an element of an ABP e.g.,MM, to be varied in the candidate ABP library is selected forrandomization. The candidate ABPs in the library can be fully randomizedor biased in their randomization, e.g. in nucleotide/residue frequencygenerally or in position of amino acid(s) within an element. By“randomized” is meant that any genetically-encodable amino acid can beprovided at any given position within a randomized amino acid sequence.An amino acid sequence of an element of an ABP that is to be optimizedcan also be partially randomized. For example, the ABP element (e.g.,candidate MM) can be partially randomized so as to provide for only asubset of amino acids at a selected position (e.g., to provide for aflexible linker at a selected position in the amino acid sequence, toprovide for an amino acid residue of a desired characteristic (e.g.,hydrophobic, polar, positively charged, negatively charged, etc.). Inanother example, the ABP element (e.g., candidate MM) can be partiallyrandomized so that one or more residues within the otherwise randomizedamino acid sequence is selected and held as invariable among apopulation or subpopulation of ABP library members (e.g., so as toprovide a cysteine at a desired position within the candidate MM).

Where the ABP is a dual target-binding ABP, a first TBM may be “fixed”and the second TBM having a known target binding activity can beprovided in its unmodified form (e.g., a native amino acid sequencehaving a known target binding activity) or can be modified (e.g., bydirected or random mutagenesis) and screened for activity in providing a“switchable” phenotype. TBMs that are identified through the screeningmethods can subsequently be evaluated for activity in binding the targetof interest, e.g., to determine that the “masking” TBM retains a desiredlevel of target binding.

Using such methods candidate ABPs having a variety of different possiblecombinations of amino acid sequence over the length of the amino acidsequence of an element(s) to be varied can be generated, thus providinga library of randomized candidate ABPs. As such, in some embodiments,the library of candidate ABPs can be fully randomized, with no sequencepreferences or constants at any position of an element(s) to beoptimized. In other embodiments, the library of candidate peptides isbiased. That is, some positions within the sequence are either heldconstant, or are selected from a limited number of possibilities. Forexample, in one embodiment, the nucleotides or amino acid residues arerandomized within a defined class, for example, of hydrophobic aminoacids, hydrophilic residues, sterically biased (either small or large)residues, towards the creation of cysteines, for cross-linking, prolinesfor SH-3 domains, serines, threonines, tyrosines or histidines forphosphorylation sites, etc., or to purines, etc.

Methods of Screening for Activatable Binding Polypeptides

The present disclosure provides methods of identifying ABPs, which canbe enzymatically activated ABPs, reducing agent-susceptible ABPs, or anABP that is activatable by either or both of enzymatic activation orreducing agent-based activation. Generally, the methods includecontacting a plurality of candidate ABPs with a target capable ofbinding a TBM of the ABPs and a protease capable of cleaving a CM of theABPs, selecting a first population of members of said plurality whichbind to the target when exposed to protease, contacting said firstpopulation with the target in the absence of the protease, and selectinga second population of members from said first population by depletingfrom said first population members that bind the target in the absenceof the protease, wherein said method provides for selection of candidateABPs which exhibit decreased binding to the target in the absence of theprotease as compared to target binding in the presence of the protease.

In general, the method for screening for candidate ABPs having a desiredswitchable phenotype is accomplished through a positive screening step(to identify members that bind target following exposure to protease)and a negative screening step (to identify members that do not bindtarget when not exposed to protease). The negative screening step can beaccomplished by, for example, depleting from the population members thatbind the target in the absence of the protease. It should be noted thatthe library screening methods described herein can be initiated byconducting the negative screening first to select for candidates that donot bind labeled target in the absence of enzyme treatment (i.e., do notbind labeled target when not cleaved), and then conducting the positivescreening (i.e., treating with enzyme and selecting for members whichbind labeled target in the cleaved state). However, for convenience, thescreening method is described below with the positive selection as afirst step.

The positive and negative screening steps can be conveniently conductedusing flow cytometry to sort candidate ABPs based on binding of adetectably labeled target. For example, as illustrated in the schematicof FIG. 18, candidate ABPs having a CM susceptible to cleavage by aprotease (such as the one exemplified in FIG. 18) can be expressed in ahost cell (e.g., E. coli) in a display scaffold (exemplified by CPX).Host cells displaying the candidate ABP are exposed to a proteasecapable of cleaving the CM and to a detectably labeled target that iscapable of binding the TBM. As illustrated in the lower panel of theright side of FIG. 18, the cells are sorted by FACS for intensity ofdetectable signal (exemplified by red fluorescence) of the detectablylabeled target. The cells that are detectably labeled include thosedisplaying a candidate ABP that was present on the cell surface,contained a CM cleavable by the protease, and that bound to detectablylabeled target. The unlabeled subpopulation (or population havingrelative lower detectable signal) represents host cells that fail tobind target at a desirable level. The “labeled” subpopulation can thenbe collected and subjected to a negative screen in which the candidateABPs are exposed to detectably labeled target in the absence ofprotease. As exemplified in the upper panel on the right side of FIG.18, those cells that are unlabeled include those that present on theirsurface a candidate ABP that has relatively lower or no detectablebinding of detectably labeled target relative to other members of thepopulation. Cells that are detectably labeled include those displaying acandidate ABP that binds target in the absence of cleavage. The“unlabeled” subpopulation can then be collected and, if desired,subjected to further rounds or screening.

One “round” or “cycle” of the screening procedure involves both apositive selection step and a negative selection step. The methods maybe repeated for a library such that multiple cycles (including completeand partial cycles, e.g., 1.5 cycles, 2.5 cycles, etc.) are performed.In this manner, members of the plurality of candidate ABPs that exhibitthe switching characteristics of an ABP may be enriched in the resultingpopulation.

In general, the screening methods are conducted by first generating anucleic acid library encoding a plurality of candidate ABPs in a displayscaffold, which is in turn introduced into a display scaffold forexpression on the surface of a replicable biological entity. As usedherein, a “plurality of candidate activatable binding polypeptides,” ora “plurality of candidate ABPs” refers to a plurality of polypeptideshaving amino acid sequences encoding candidate ABPs, where members ofthe plurality are variable with respect to the amino acid sequence of atleast one of the components of an ABP, e.g., the plurality is variablewith respect to the amino acid sequence of the MM, the CM or the TBM,usually the MM.

For example, the TBM and CM of the candidate ABPs are held “fixed” andthe candidate ABPs in the library are variable with respect to the aminoacid sequence of the MM. The variable amino acid sequence of the MM isreferred to hereinafter as a candidate masking moiety (candidate MM). Asillustrated in FIG. 19, libraries can be generated having different MMs,which can include, for example, candidate ABPs having an MM that isdesigned to position a cysteine residue to “force” formation of adisulfide bond with another cysteine present in the candidate ABP (withother residues selected to provide an MM having an amino acid sequencethat is otherwise fully or at least partially randomized). In anotherexample, a library can be generated to include candidate ABPs having anMM that is designed to position a cysteine residue such that disulfidebond formation with another cysteine in the candidate ABP is favored(with other residues selected to provide an MM having an amino acidsequence that is otherwise fully or at least partially randomized). Inanother example, a library can be generated to include candidate ABPs inwhich the MM includes a fully randomized amino acid sequence. Suchlibraries can contain candidate ABPs designed by one or more of thesecriterion. By screening members of said plurality according to themethods described herein, members having candidate MMs that provide adesired switchable phenotype can be identified.

The term “candidate”, as used in the context of, for example, “candidateABP” or “candidate MM” (or other element of an ABP that is to bescreened), refers to a polypeptide that is to be screened to determinewhether it exhibits desired structural and/or functionalcharacteristics. For example, a “candidate ABP” refers to a polypeptidethat is designed to resemble the structure of an ABP as describedherein, except that at least one of the MM, CM, TBM, and linker(s) arevariable with respect to their amino acid sequences, wherein thecandidate ABP is to be screened for a desired switchable phenotype. A“candidate MM”, for example, refers to an amino acid sequence of an ABPwhich is to be screened for its function as a masking moiety in thecontext of an ABP.

In one embodiment of the methods, each member of the plurality ofcandidate ABPs is displayed on the surface of a replicable biologicalentity (exemplified here by bacterial cells). The members of theplurality are exposed to a protease capable of cleaving the CM of thecandidate ABPs and contacted with a target which is a binding partner ofthe TBM of the candidate ABPs. Bacterial cells displaying memberscomprising TBMs which bind the target after exposure to the protease areidentified and/or separated via detection of target binding (e.g.,detection of a target-TBM complex). Members comprising TBMs which bindthe target after protease exposure (which can lead to cleavage of theCM) are then contacted with the target in the absence of the protease.Bacterial cells displaying members comprising TBMs which exhibitdecreased or undetectable binding to the target in the absence ofcleavage are identified and/or separated via detection of cells lackingbound target. In this manner, members of the plurality of candidate ABPswhich bind target in a cleaved state and exhibit decreased orundetectable target binding in an uncleaved state are identified and/orselected.

As noted above, candidate ABP libraries can be constructed so as toscreen for one or more aspects of the ABP constructs, e.g., to providefor optimization of a switchable phenotype for one or more of the MM,the CM, and the TBM. One or more other elements of the ABP can be variedto facilitate optimization. For example: vary the MM, including varyingthe number or position of cysteines or other residues that can providefor different conformational characteristics of the ABP in the absenceof cleaving agent (e.g., enzyme): vary the CM to identify a substratethat is optimized for one or more desired characteristics (e.g.,specificity of enzyme cleavage, and the like); and/or vary the TBM toprovide for optimization of “switchable” target binding.

In general, the elements of the candidate ABP libraries are selectedaccording to a target protein of interest, where the ABP is to beactivated to provide for enhanced binding of the target in the presenceof a cleaving agent (e.g., enzyme) that cleaves the CM. For example,where the CM and TBM are held “fixed” among the library members, the CMis selected such that it is cleavable by a cleaving agent (e.g., enzyme)that is co-localized with a target of interest, where the target ofinterest is a binding partner of the TBM. In this manner, an ABP can beselected such that it is selectively activated under the appropriatebiological conditions, and thus at an appropriate biological location.For example, where it is desired to develop an ABP to be used as ananti-angiogenic compound and exhibit a switchable phenotype for VEGFbinding, the CM of the candidate ABP is selected to be a substrate foran enzyme and/or a reducing agent that is colocalized with VEGF (e.g., aCM cleavable by a matrix-metalloprotease).

As discussed above, a TBM is generally selected according to a target ofinterest. Many targets are known in the art. Biological targets ofinterest include protein targets that have been identified as playing arole in disease. Such targets include but are not limited to cellsurface receptors and secreted binding proteins (e.g., growth factors),soluble enzymes, structural proteins (e.g. collagen, fibronectin) andthe like. Exemplary non-limiting targets are presented in Table 1, butother suitable targets will be readily identifiable by those of ordinaryskill in the art. In addition, many proteases are known in the art whichco-localize with targets of interest. As such, persons of ordinary skillin the art will be able to readily identify appropriate enzymes andenzyme substrates for use in the above methods.

Optional Enrichment for Cell Surface Display Prior to ABP Screening

Prior to the screening method, it may be desirable to enrich for cellsexpressing an appropriate peptide display scaffold on the cell surface.The optional enrichment allows for removal of cells from the celllibrary that (1) do not express peptide display scaffolds on the cellouter membrane or (2) express non-functional peptide display scaffoldson the cell outer membrane. By “non-functional” is meant that thepeptide display scaffold does not properly display a candidate ABP,e.g., as a result of a stop codon or a deletion mutation.

Enrichment for cells can be accomplished by growing the cell populationand inducing expression of the peptide display scaffolds. The cells arethen sorted based on, for example, detection of a detectable signal ormoiety incorporated into the scaffold or by use of a detectably-labeledantibody that binds to a shared portion of the display scaffold or theABP. These methods are described in greater detail in U.S. PatentApplication Publication No: 2007/0065878, published Mar. 22, 2007.

Screening for Target Binding by Cleaved ABPs

Prior to screening, the candidate ABP library can be expanded (e.g., bygrowth in a suitable medium in culture under suitable conditions).Subsequent to the optional expansion, or as an initial step, the libraryis subjected to a first screen to identify candidate ABPs that bindtarget following exposure to protease. Accordingly, this step is oftenreferred to herein as the “positive” selection step.

In order to identify members that bind target following proteasecleavage, the candidate ABP library is contacted with a protease capableof cleaving the CM of the displayed candidate ABPs for an amount of timesufficient and under conditions suitable to provide for cleavage of theprotease substrate of the CM. A variety of protease-CM combinations willbe readily ascertainable by those of ordinary skill in the art, wherethe protease is one which is capable of cleaving the CM and one whichco-localizes in vivo with a target of interest (which is a bindingpartner of the TBM). For example, where the target of interest is asolid tumor associated target (e.g. VEGF), suitable enzymes include, forexample, Matrix-Metalloproteases (e.g., MMP-2), A Disintegrin andMetalloprotease(s) (ADAMs)/ADAM with thrombospondin-like motifs(ADAMTS), Cathepsins and Kallikreins. The amino acid sequences ofsubstrates useful as CMs in the ABPs described herein are known in theart and, where desired, can be screened to identify optimal sequencessuitable for use as a CM by adaptation of the methods described herein.Exemplary substrates can include but are not limited to substratescleavable by one or more of the following enzymes: MMP-1, MMP-2, MMP-3,MMP-8, MMP-9, MMP-14, PLASMIN, PSA, PSMA, CATHEPSIN D, CATHEPSIN K,CATHEPSIN S, ADAM10, ADAM12, ADAMTS, Caspase-1, Caspase-2, Caspase-3,Caspase-4, Caspase-5, Caspase-6, Caspase-7, Caspase-8, Caspase-9,Caspase-10, Caspase-11, Caspase-12, Caspase-13, Caspase-14, and TACE.

The candidate ABP library is also exposed to target for an amount oftime sufficient and under conditions suitable for target binding, whichconditions can be selected according to conditions under which targetbinding to the TBM would be expected. The candidate ABP library can beexposed to the protease prior to exposure to target (e.g., to provide apopulation of candidate ABPs which include cleaved ABPs) or incombination with exposure to target, usually the latter so as to bestmodel the expected in vivo situation in which both protease and targetwill be present in the same environmental milieu. Following exposure toboth protease and target, the library is then screened to select membershaving bound target, which include candidate ABPs in a target-TBMcomplex.

Detection of target-bound candidate ABPs can be accomplished in avariety of ways. For example, the target may be detectably labeled andthe first population of target-bound candidate ABPs may be selected bydetection of the detectable label to generate a second population havingbound target (e.g., a positive selection for target-bound candidateABPs).

Screening for Candidate ABPs that do not Bind Target in the Absence ofProtease Cleavage

The population of candidate ABPs selected for target binding followingexposure to protease can then be expanded (e.g., by growth in a suitablemedium in culture under suitable conditions), and the expanded librarysubjected to a second screen to identify members exhibiting decreased orno detectable binding to target in the absence of protease exposure. Thepopulation resulting from this second screen will include candidate ABPsthat, when uncleaved, do not bind target significantly or to adetectable level. Accordingly, this step is often referred to herein asthe “negative” selection step.

The population that resulted from the first screen is contacted withtarget in the absence of the protease for a time sufficient and underconditions suitable for target binding, which conditions can be selectedaccording to conditions under which target binding to the TBM would beexpected. A negative selection can then be performed to identifycandidate ABPs that are relatively decreased for target binding,including those which exhibit no detectably target binding. Thisselection can be accomplished by, for example, use of a detectablylabeled target, and subjecting the target-exposed population to flowcytometry analysis to sort into separate subpopulation those cells thatdisplay a candidate ABP that exhibits no detectable target bindingand/or which exhibit a relatively lower detectable signal. Thissubpopulation is thus enriched for cells having a candidate ABP thatexhibit decreased or undetectable binding to target in the absence ofcleavage.

Detectable Labels

As used herein, the terms “label”, “detectable label” and “detectablemoiety” are used interchangeably to refer to a molecule capable ofdetection, including, but not limited to, radioactive isotopes,fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions,metal sols, ligands (e.g., biotin, avidin, strepavidin or haptens) andthe like. The term “fluorescer” refers to a substance or a portionthereof which is capable of exhibiting fluorescence in the detectablerange. Exemplary detectable moieties suitable for use as target labelsinclude, affinity tags and fluorescent proteins.

The term “affinity tag” is used herein to denote a peptide segment thatcan be attached to a target that can be detected using a molecule thatbinds the affinity tag and provides a detectable signal (e.g., afluorescent compound or protein). In principal, any peptide or proteinfor which an antibody or other specific binding agent is available canbe used as an affinity tag. Exemplary affinity tags suitable for useinclude, but are not limited to, a monocytic adaptor protein (MONA)binding peptide, a T7 binding peptide, a streptavidin binding peptide, apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)), orother antigenic epitope or binding domain. See, in general, Ford et al.,Protein Expression and Purification 2:95 (1991). DNA molecules encodingaffinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.).

Any fluorescent polypeptide (also referred to herein as a fluorescentlabel) well known in the art is suitable for use as a detectable moietyor with an affinity tag of the peptide display scaffolds describedherein. A suitable fluorescent polypeptide will be one that can beexpressed in a desired host cell, such as a bacterial cell or amammalian cell, and will readily provide a detectable signal that can beassessed qualitatively (positive/negative) and quantitatively(comparative degree of fluorescence). Exemplary fluorescent polypeptidesinclude, but are not limited to, yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), GFP, mRFP, RFP (tdimer2), HCRED, etc., or anymutant (e.g., fluorescent proteins modified to provide for enhancedfluorescence or a shifted emission spectrum), analog, or derivativethereof. Further suitable fluorescent polypeptides, as well as specificexamples of those listed herein, are provided in the art and are wellknown.

Biotin-based labels also find use in the methods disclosed herein.Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see, e.g.,chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, herebyincorporated by reference. A biotinylated substrate can be detected bybinding of a detectably labeled biotin binding partner, such as avidinor streptavidin. Similarly, a large number of haptenylation reagents arealso known.

Screening Methods

Any suitable method that provides for separation and recovery of ABPs ofinterest may be utilized. For example, a cell displaying an ABP ofinterest may be separated by FACS, immunochromatography or, where thedetectable label is magnetic, by magnetic separation. As a result of theseparation, the population is enriched for cells that exhibit thedesired characteristic, e.g., exhibit binding to target followingcleavage or have decreased or no detectable binding to target in theabsence of cleavage.

For example, selection of candidate ABPs having bound detectably labeledtarget can be accomplished using a variety of techniques known in theart. For example, flow cytometry (e.g., FACS®) methods can be used tosort detectably labeled candidate ABPs from unlabeled candidate ABPs.Flow cyomtery methods can be implemented to provide for more or lessstringent requirements in separation of the population of candidateABPs, e.g., by modification of gating to allow for “dimmer” or torequire “brighter” cell populations in order to be separated into thesecond population for further screening.

In another example, immunoaffinity chromatography can be used toseparate target-bound candidate ABPs from those that do not bind target.For example, a support (e.g., column, magnetic beads) having boundanti-target antibody can be contacted with the candidate ABPs that havebeen exposed to protease and to target. Candidate ABPs having boundtarget bind to the anti-target antibody, thus facilitating separationfrom candidate ABPs lacking bound target. Where the screening step is toprovide for a population enriched for uncleaved candidate ABPs that haverelatively decreased target binding or no detectable target binding(e.g., relative to other candidate ABPs), the subpopulation of interestis those members that lack or have a relatively decreased detectablysignal for bound target. For example, where an immunoaffinity techniqueis used in such negative selection for bound target, the subpopulationof interest is that which is not bound by the anti-target support.

Screening for Dual Target-Binding ABPs

Methods for screening disclosed herein can be readily adapted toidentify dual target-binding ABPs having a desired switchable phenotypedue to interaction between two TBMs. In general, rather than a candidateMM in the example above, a TBM having a known binding activity ispresented in the candidate ABP in place of the MM. In general, themethod thus involves a library containing a plurality of candidate ABPs,wherein each member of said plurality comprises a first TBM, a secondTBM and a CM. The library is contacted with target capable of binding atleast the first TBM and a cleaving agent capable of cleaving the CM. Afirst population of members of the library is selected for binding thetarget in the presence of the cleaving agent (e.g., protease for theCM). This selected population is then subjected to the negative screenabove, in which binding of target to the library members in the absenceof the cleaving agent is assessed. A second population of members isthen generated by depleting the subpopulation of members that bind tosaid target in the absence of the cleaving agent. This can beaccomplished by, for example, sorting members that are not bound totarget away from those that are bound to target, as determined bydetection of a detectably labeled target. This method thus provides forselection of candidate ABPs which exhibit decreased binding to thetarget in the absence of the cleaving agent as compared to binding tosaid target in the presence of the cleaving agent.

This method can be repeated for both targets, although target binding toa TBM that is not associated with a display scaffold following cleavagemust be assessed by evaluating the presence or absence (and/or relativelevels) of target complexed with TBM-containing ABP fragments insolution.

In one example, a library containing a plurality of candidate ABPs isgenerated, wherein each member comprises a first TBM, a second TBM and aCM, where the CM is positioned between the first and second TBMs, andwhere the first TBM is immobilized on the surface of a replicablebiological entity via a display scaffold. The library is then subjectedto the positive and negative screening steps above with a target that iscapable of binding the second TBM. For example, the library is contactedwith a target capable of binding the first TBM and a cleaving agentcapable of cleaving the CM, then selecting a first population of membersof said plurality which bind to said target in the presence of thecleaving agent. The selected first population is then contacted withtarget capable of binding the first TBM in the absence of the cleavingagent, and a second population of members selected that bind to saidtarget in the absence of the cleaving agent.

As above, any element of a dual target binding ABP can be varied withinthe library. For example, a first TBM may be “fixed” and the second TBMhaving a known target binding activity can be provided in its unmodifiedform (e.g., a native amino acid sequence having a known target bindingactivity) or can be modified (e.g., by directed or random mutagenesis)and screened for activity in providing a “switchable” phenotype. TBMsthat are identified through the screening methods as exhibiting“masking” activity can subsequently be evaluated for activity in bindingthe target of interest, e.g., to determine that the “masking” TBMretains a desired level of target binding. For example, a constructencoding a display scaffold and a TBM-MM dual function element of a dualtarget-binding ABP can be inserted for expression and display on areplicable biological entity surface. Binding of the TBM-MM can then beevaluated according to methods in the art.

Exemplary Variations of the Screening Methods to Select for CandidateABPs

The above method may be modified to select for populations and librarymembers that demonstrate desired switching characteristics.

Iterative Screens to Identify and/or Optimize ABP Elements

The methods and candidate ABP libraries described herein can be readilyadapted to provide for identification and/or optimization of one or moreelements of an ABP. For example, candidate ABPs that vary with respectto any one or more of TBM, CM, linkers, and the like can be produced andsubjected to the screening methods described herein.

Reducing Agent-Activatable ABPs

While the methods above describe screening methods for identifying ABPS,it should be understood that an ABP or candidate ABP with a CM that canfacilitate formation of a cysteine-cysteine disulfide bond in an ABP canalso be subjected to the screening methods disclosed herein. Such ABPsmay or may not further include a CM (which may be the same or differentCM) that may or may not comprise a protease substrate. In theseembodiments, the positive screen described above may be conducted byexposing an ABP or candidate ABP to a reducing agent (e.g., to reducingconditions) capable of cleaving the disulfide bond of thecysteine-cysteine pair of the ABP. The negative screen can then beconducted in the absence of the reducing conditions. As such, a libraryproduced having may be enriched for ABPs which are activatable byexposure to disulfide bond reducing conditions.

Number of Cycles and Scaffold Free Screening of ABPs

By increasing the number of cycles of the above methods, populations andlibrary members that demonstrate improved switching characteristics canbe identified, as exemplified in FIG. 8. Any number of cycles ofscreening can be performed.

In addition, individual clones of candidate ABPs can be isolated andsubjected to screening so as to determine the dynamic range of thecandidate ABP. Candidate ABPs can also be tested for a desiredswitchable phenotype separate from the scaffold, i.e., the candidate ABPcan be expressed or otherwise generated separate from the displayscaffold, and the switchable phenotype of the candidate ABP assessed inthe absence of the scaffold and, where desired, in a cell-free system(e.g., using solubilized ABP).

It may be desirable to provide an ABP expression construct whichincludes a secretion signal to provide for extracellular secretion ofthe ABP, thereby facilitating production and recovery of an ABP ofinterest. Secretion signals suitable for use in bacterial and mammaliansystems are well known in the art.

Optimization of ABP Components and Switching Activity

The above methods may be modified to optimize the performance of an ABP,e.g., an ABP identified in the screening method described herein. Forexample, where it is desirable to optimize the performance of themasking moiety, e.g., to provide for improved inhibition of targetbinding of the TBM in the uncleaved state, the amino acid sequences ofthe TBM and the CM may be “fixed” in a candidate ABP library, and the MMvaried such that members of a library have variable MMs relative to eachother. The MM may be optimized in a variety of ways including alterationin the number and or type of amino acids that make up the MM. Forexample, each member of the plurality of candidate ABPs may comprise acandidate MM, wherein the candidate MM comprises at least one cysteineamino acid residue and the remaining amino acid residues are variablebetween the members of the plurality. In a further example, each memberof the plurality of candidate ABPs may comprise a candidate MM, whereinthe candidate MM comprises a cysteine amino acid residue and a randomsequence of amino acid residues, e.g., a random sequence of 5 aminoacids.

Selection for Expanded Dynamic Range

As noted above, ABPs having a desired dynamic range with respect totarget binding in the cleaved versus uncleaved state are of particularinterest. Such ABPs are those that, for example, have no detectablebinding in the presence of target at physiological levels found attreatment and non-treatment sites in a subject but which, once cleavedby protease, exhibit high affinity and/or high avidity binding totarget. The greater the dynamic range of an ABP, the better the“switchable” phenotype of the ABP. Thus ABPs can be optimized to selectfor those having an “expanded” dynamic range for target binding in thepresence and absence of a cleaving agent.

The screening methods described herein can be modified so as to enhanceselection of ABPs having a desired and/or optimized dynamic range. Ingeneral, this can be accomplished by altering the concentrations oftarget utilized in the positive selection and negative selection stepsof the method such that screening for target binding of ABPs exposed toprotease (i.e., the screening population that includes cleaved ABPs) isperformed using a relatively lower target concentration than whenscreening for target binding of uncleaved ABPs. Accordingly, the targetconcentration is varied between the steps so as to provide a “selectivepressure” toward a switchable phenotype. Where desired, the differencein target concentrations used at the positive and negative selectionsteps can be increased with increasing cycle number.

Use of a relatively lower concentration of target in the positiveselection step can serve to drive selection of those ABP members thathave improved target binding when in the cleaved state. For example, thescreen involving protease-exposed ABPs can be performed at a targetconcentration that is from about 2 to about 100 fold lower, about 2 to50 fold lower, about 2 to 20 fold lower, about 2 to 10-fold lower, orabout 2 to 5-folder lower than the Kd of the TBM-target interaction. Asa result, after selection of the population for target-bound ABPs, theselected population will be enriched for ABPs that exhibit higheraffinity and/or avidity binding relative to other ABPs in thepopulation.

Use of a relatively higher concentration of target in the negativeselection step can serve to drive selection of those ABP members thathave decreased or no detectable target binding when in the uncleavedstate. For example, the screen involving ABPs that have not been exposedto protease (in the negative selection step) can be performed at atarget concentration that is from about 2 to about 100 fold higher,about 2 to 50 fold higher, about 2 to 20 fold higher, about 2 to 10-foldhigher, or about 2 to 5-folder higher, than the Kd of the TBM-targetinteraction. As a result, after selection of the population for ABPsthat do not detectably bind target, the selected population will beenriched for ABPs that exhibit lower binding for target when in theuncleaved state relative to other uncleaved ABPs in the population.Stated differently, after selection of the population for ABPs that donot detectably bind target, the selected population will be enriched forABPs for which target binding to TBM is inhibited, e.g., due to“masking” of the TBM from target binding.

Where the ABP is a dual target-binding ABP, the screening methoddescribed above can be adapted to provide for ABPs having a desireddynamic range for a first target that is capable of binding a first TBMand for a second target that is capable of binding a second TBM. Targetbinding to a TBM that is located on a portion of the ABP that is“cleaved away” from the ABP presented on a display scaffold can beevaluated by assessing formation of target-TBM complexes in solution(e.g., in the culture medium), e.g., immunochromatography having ananti-ABP fragment antibody to capture cleaved fragment, then detectingbound, detectably labeled target captured on the column.

Testing of Soluble ABPs

Candidate ABPs can be tested for their ability to maintain a“switchable” phenotype while in soluble form. One such method involvesthe immobilization of target to support (e.g., an array, e.g., aBiacore™ CM5 sensor chip surface). Immobilization of a target ofinterest can be accomplished using any suitable techniques (e.g.,standard amine coupling). The surface of the support can be blocked toreduce non-specific binding. Optionally, the method can involve use of acontrol (e.g., a support that does not contain immobilized target (e.g.,to assess background binding to the support) and/or contains a compoundthat serves as a negative control (e.g., to assess specificity ofbinding of the candidate ABP to target versus non-target).

After the target is covalently immobilized, the candidate ABP iscontacted with the support under conditions suitable to allow forspecific binding to immobilized target. The candidate ABP can becontacted with the support-immobilized target in the presence and in theabsence of a suitable cleavage agent in order to assess the “switchable”phenotype. Assessment of binding of the candidate ABP in the presence ofcleavage agent as compared to in the absence of cleavage agent and,optionally, compared to binding in a negative control provides a bindingresponse, which in turn is indicative of the “switchable” phenotype.

Screening for Individual Moieties for Use in Candidate ABPs

It may be desirable to screen separately for one or more of the moietiesof a candidate ABP, e.g., a TBM, MM or CM, prior to testing thecandidate ABP for a “switchable” phenotype. For example, known methodsof identifying peptide substrates cleavable by specific proteases can beutilized to identify cleavable moieties for use in ABPs designed foractivation by such proteases. In addition a variety of methods areavailable for identifying peptide sequences which bind to a target ofinterest. These methods can be used, for example, to identify TBMs whichbinds to a particular target or to identify a MM which binds to aparticular TBM.

The above methods include, for example, methods in which a moiety of acandidate ABP, e.g., a TBM, MM or CM, is displayed using a replicablebiological entity.

As discussed previously herein, a variety of display technologies usingreplicable biological entities are known in the art. These methods andentities include, but are not limited to, display methodologies such asmRNA and ribosome display, eukaryotic virus display, and bacterial,yeast, and mammalian cell surface display. See Wilson, D. S., et al.2001 PNAS USA 98(7):3750-3755; Muller, O. J., et al. (2003) Nat.Biotechnol. 3:312; Bupp, K. and M. J. Roth (2002) Mol. Ther. 5(3):3293513; Georgiou, G., et al., (1997) Nat. Biotechnol. 15(1):29 3414; andBoder, E. T. and K. D. Wittrup (1997) Nature Biotech. 15(6):553 557.Surface display methods are attractive since they enable application offluorescence-activated cell sorting (FACS) for library analysis andscreening. See Daugherty, P. S., et al. (2000) J. Immuunol. Methods243(1 2):211 2716; Georgiou, G. (2000)Adv. Protein Chem. 55:293 315;Daugherty, P. S., et al. (2000) PNAS USA 97(5):2029 3418; Olsen, M. J.,et al. (2003) Methods Mol. Biol. 230:329 342; Boder, E. T. et al. (2000)PNAS USA 97(20):10701 10705; Mattheakis, L. C., et al. (1994) PNAS USA91(19): 9022 9026; and Shusta, E. V., et al. (1999) Curr. Opin. Biotech.10(2):117 122. Additional display methodologies which may be used toidentify a peptide capable of binding to a biological target of interestare described in U.S. Pat. No. 7,256,038, the disclosure of which isincorporated herein by reference.

Phage display involves the localization of peptides as terminal fusionsto the coat proteins, e.g., pIII, pIIV of bacteriophage particles. SeeScott, J. K. and G. P. Smith (1990) Science 249(4967):386 390; andLowman, H. B., et al. (1991) Biochem. 30(45):10832 10838. Generally,polypeptides with a specific function of binding are isolated byincubating with a target, washing away non-binding phage, eluting thebound phage, and then re-amplifying the phage population by infecting afresh culture of bacteria.

Exemplary phage display and cell display compositions and methods aredescribed in U.S. Pat. Nos. 5,223,409; 5,403,484; 7,118,879; 6,979,538;7,208,293; 5571698; and 5,837,500.

Additional exemplary display scaffolds and methods include thosedescribed in U.S. Patent Application Publication No: 2007/0065878,published Mar. 22, 2007.

Optionally, the display scaffold can include a protease cleavage site(different from the protease cleavage site of the CM) to allow forcleavage of an ABP or candidate ABP from a surface of a host cell.

In one, where the replicable biological entity is a bacterial cell,suitable display scaffolds include circularly permuted Esccherichia coliouter membrane protein OmpX (CPX) described by Rice et al, Protein Sci.(2006) 15: 825-836. See also, U.S. Pat. No. 7,256,038, issued Aug. 14,2007.

Automated Screening Methods

The screening methods described herein may be automated to provideconvenient, real time, high volume methods of screening a library ofABPs for a desired switchable activity. Automated methods can bedesigned to provide for iterative rounds of positive and negativeselection, with the selected populations being separated andautomatically subjected to the next screen for a desired number ofcycles.

Analysis points to assess of candidate ABPs in a population may be overtime following completion of a positive selection step, a negativeselection step, or both. In addition, information regarding the averagedynamic range of a population of candidate ABPs at selected targetconcentrations in the positive and negative selection steps can bemonitored and stored for later analysis, e.g. so as to assess the effectof selective pressure of the different target concentrations.

A computer program product can control operation of the detection and/ormeasuring means and can perform numerical operations relating to theabove-described steps, and generate a desired output (e.g., flowcytometry analysis, etc.). Computer program product comprises a computerreadable storage medium having computer-readable program code meansembodied in the medium. Hardware suitable for use in such automatedapparatus will be apparent to those of skill in the art, and may includecomputer controllers, automated sample handlers, fluorescencemeasurement tools, printers and optical displays. The measurement toolmay contain one or more photodetectors for measuring the fluorescencesignals from samples where fluorescently detectable molecules areutilized. The measurement tool may also contain a computer-controlledstepper motor so that each control and/or test sample can be arranged asan array of samples and automatically and repeatedly positioned oppositea photodetector during the step of measuring fluorescence intensity.

The measurement tool (e.g., a fluorescence activated cell sorter) can beoperatively coupled to a general purpose or application specificcomputer controller. The controller can comprise a computer programproduce for controlling operation of the measurement tool and performingnumerical operations relating to the above-described steps. Thecontroller may accept set-up and other related data via a file, diskinput or data bus. A display and printer may also be provided tovisually display the operations performed by the controller. It will beunderstood by those having skill in the art that the functions performedby the controller may be realized in whole or in part as softwaremodules running on a general purpose computer system. Alternatively, adedicated stand-alone system with application specific integratedcircuits for performing the above described functions and operations maybe provided.

Methods of Use of ABPs in Therapy

ABPs can be incorporated into pharmaceutical compositions containing,for example, a therapeutically effective amount of an ABP of interestand a carrier pharmaceutically acceptable excipient (also referred to asa pharmaceutically acceptable carrier). Many pharmaceutically acceptableexcipients are known in the art, are generally selected according to theroute of administration, the condition to be treated, and other suchvariables that are well understood in the art. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7^(th) ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3^(rd) ed. Amer. Pharmaceutical Assoc. Pharmaceutical compositions canalso include other components such as pH adjusting and buffering agents,tonicity adjusting agents, stabilizers, wetting agents and the like. Insome embodiments, nanoparticles or liposomes carry a pharmaceuticalcomposition comprising an ABP.

Suitable components for pharmaceutical compositions of ABPs can beguided by pharmaceutical compositions that may be available for a TBM ofthe ABP. For example, where the, the ABP include a VEGF binder (i.e.,VEGF inhibitor), such ABPs can be formulated in a pharmaceuticalformulation according to methods and compositions suitable for use withthe VEGF binder. In embodiments where the ABP comprises a full lengthantibody or an antigen binding fragment thereof, the composition can beformulated in a pharmaceutical formulation according to methods andcompositions suitable for use with antibodies and antigen bindingfragments.

In general, pharmaceutical formulations of one or more ABPs are preparedfor storage by mixing the ABP having a desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes. Pharmaceutical formulations may also contain more than oneactive compound as necessary for the particular indication beingtreated, where the additional active compounds generally are those withactivities complementary to an ABP. Such compounds are suitably presentin combination in amounts that are effective for the purpose intended.

The pharmaceutical formulation can be provided in a variety of dosageforms such as a systemically or local injectable preparation. Thecomponents can be provided in a carrier such as a microcapsule, e.g.,such as that prepared by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations are also within the scope ofABP-containing formulations. Exemplary sustained-release preparationscan include semipermeable matrices of solid hydrophobic polymerscontaining the antibody, which matrices are in the form of shapedarticles, e.g., films, or microcapsule. Examples of sustained-releasematrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated ABPs remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37 degreesC., resulting in decreased biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be undesirable intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

ABPs can be conjugated to delivery vehicles for targeted delivery of anactive agent that serves a therapeutic purpose. For example, ABPs can beconjugated to nanoparticles or liposomes having drugs encapsulatedtherein or associated therewith. In this manner, specific, targeteddelivery of the drug can be achieved. Methods of linking polypeptides toliposomes are well known in the art and such methods can be applied tolink ABPs to liposomes for targeted and or selective delivery ofliposome contents. By way of example, polypeptides can be covalentlylinked to liposomes through thioether bonds. PEGylated gelatinnanoparticles and PEGylated liposomes have also been used as a supportfor the attachment of polypeptides, e.g., single chain antibodies. See,e.g., Immordino et al. (2006) Int J Nanomedicine. September; 1(3):297-315, incorporated by reference herein for its disclosure of methodsof conjugating polypeptides, e.g., antibody fragments, to liposomes.

Methods of Treatment

ABPs described herein can be selected for use in methods of treatment ofsuitable subjects according to the CM-TBM combination provided in theABP. Examples based on the VEGF-inhibiting ABP are provided below.

The ABP can be administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, ifdesired for local injection (e.g., at the site of a solid tumor).Parenteral administration routes include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration.

The appropriate dosage of ABP will depend on the type of disease to betreated, the severity and course of the disease, the patient's clinicalhistory and response to the ABP, and the discretion of the attendingphysician. ABPs can suitably be administered to the patient at one timeor over a series of treatments.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of ABP can serve as an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on factors such as those mentioned herein. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful.

The ABP composition will be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe ABP, the method of administration, the scheduling of administration,and other factors known to medical practitioners. The “therapeuticallyeffective amount” of an ABP to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat a disease or disorder.

Generally, alleviation or treatment of a disease or disorder involvesthe lessening of one or more symptoms or medical problems associatedwith the disease or disorder. For example, in the case of cancer, thetherapeutically effective amount of the drug can accomplish one or acombination of the following: reduce the number of cancer cells; reducethe tumor size; inhibit (i.e., to decrease to some extent and/or stop)cancer cell infiltration into peripheral organs; inhibit tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Insome embodiments, a composition of this invention can be used to preventthe onset or reoccurrence of the disease or disorder in a subject ormammal.

ABPs can be used in combination (e.g., in the same formulation or inseparate formulations) with one or more additional therapeutic agents ortreatment methods (“combination therapy”). An ABP can be administered inadmixture with another therapeutic agent or can be administered in aseparate formulation. Therapeutic agents and/or treatment methods thatcan be administered in combination with an ABP, and which are selectedaccording to the condition to be treated, include surgery (e.g.,surgical removal of cancerous tissue), radiation therapy, bone marrowtransplantation, chemotherapeutic treatment, certain combinations of theforegoing, and the like.

Use of ABPs that Inhibit VEGF in Anti-Angiogenic Therapies

Where the ABP contains a TBM that is a VEGF inhibitor, the ABP finds usein treatment of conditions in which inhibition of angiogenesis isdesired, particularly those conditions in which inhibition of VEGF is ofinterest. VEGF-inhibiting ABPs can include dual target binding ABPshaving a TBM that binds to VEGF as well as a TBM that binds to a secondgrowth factor, such as a fibroblast growth factor (e.g., FGF-2), andinhibits FGF activity. Such dual target binding ABPs thus can bedesigned to provide for inhibition of two angiogenesis-promotingfactors, and which are activatable by a cleaving agent (e.g., enzyme,such as a MMP or other enzyme as discussed herein) which co-localizes ata site of aberrant angiogenesis.

Angiogenesis-inhibiting ABPs find use in treatment of solid tumors in asubject (e.g., human), particularly those solid tumors that have anassociated vascular bed that feeds the tumor such that inhibition ofangiogenesis can provide for inhibition or tumor growth. Anti-VEGF-basedanti-angiogenesis ABPs also find use in other conditions having one ormore symptoms amenable to therapy by inhibition of abnormalangiogenesis.

In general, abnormal angiogenesis occurs when new blood vessels eithergrow excessively, insufficiently or inappropriately (e.g., the location,timing or onset of the angiogenesis being undesired from a medicalstandpoint) in a diseased state or such that it causes a diseased state.Excessive, inappropriate or uncontrolled angiogenesis occurs when thereis new blood vessel growth that contributes to the worsening of thediseased state or causes a diseased state, such as in cancer, especiallyvascularized solid tumors and metastatic tumors (including colon, lungcancer (especially small-cell lung cancer), or prostate cancer),diseases caused by ocular neovascularisation, especially diabeticblindness, retinopathies, primarily diabetic retinopathy or age-inducedmacular degeneration and rubeosis; psoriasis, psoriatic arthritis,haemangioblastoma such as haemangioma; inflammatory renal diseases, suchas glomerulonephritis, especially mesangioproliferativeglomerulonephritis, haemolytic uremic syndrome, diabetic nephropathy orhypertensive neplirosclerosis; various imflammatory diseases, such asarthritis, especially rheumatoid arthritis, inflammatory bowel disease,psorsasis, sarcoidosis, arterial arteriosclerosis and diseases occurringafter transplants, endometriosis or chronic asthma and other conditionsthat will be readily recognized by the ordinarily skilled artisan. Thenew blood vessels can feed the diseased tissues, destroy normal tissues,and in the case of cancer, the new vessels can allow tumor cells toescape into the circulation and lodge in other organs (tumormetastases).

ABP-based anti-angiogenesis therapies can also find use in treatment ofgraft rejection, lung inflammation, nephrotic syndrome, preeclampsia,pericardial effusion, such as that associated with pericarditis, andpleural effusion, diseases and disorders characterized by undesirablevascular permeability, e.g., edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion, pleural effusion, permeabilityassociated with cardiovascular diseases such as the condition followingmyocardial infarctions and strokes and the like.

Other angiogenesis-dependent diseases that may be treated usinganti-angiogenic ABPs as described herein include angiofibroma (abnormalblood of vessels which are prone to bleeding), neovascular glaucoma(growth of blood vessels in the eye), arteriovenous malformations(abnormal communication between arteries and veins), nonunion fractures(fractures that will not heal), atherosclerotic plaques (hardening ofthe arteries), pyogenic granuloma (common skin lesion composed of bloodvessels), scleroderma (a form of connective tissue disease), hemangioma(tumor composed of blood vessels), trachoma (leading cause of blindnessin the third world), hemophilic joints, vascular adhesions andhypertrophic scars (abnormal scar formation).

Amounts of ABP for administration to provide a desired therapeuticeffect will vary according to a number of factors such as thosediscussed above. In general, in the context of cancer therapy, atherapeutically effective amount of an ABP is an amount that that iseffective to inhibit angiogenesis, and thereby facilitate reduction of,for example, tumor load, atherosclerosis, in a subject by at least about5%, at least about 10%, at least about 20%, at least about 25%, at leastabout 50%, at least about 75%, at least about 85%, or at least about90%, up to total eradication of the tumor, when compared to a suitablecontrol. In an experimental animal system, a suitable control may be agenetically identical animal not treated with the agent. Innon-experimental systems, a suitable control may be the tumor loadpresent before administering the agent. Other suitable controls may be aplacebo control.

Whether a tumor load has been decreased can be determined using anyknown method, including, but not limited to, measuring solid tumor mass;counting the number of tumor cells using cytological assays;fluorescence-activated cell sorting (e.g., using antibody specific for atumor-associated antigen) to determine the number of cells bearing agiven tumor antigen; computed tomography scanning, magnetic resonanceimaging, and/or x-ray imaging of the tumor to estimate and/or monitortumor size; measuring the amount of tumor-associated antigen in abiological sample, e.g., blood or serum; and the like.

In some embodiments, the methods are effective to reduce the growth rateof a tumor by at least about 5%, at least about 10%, at least about 20%,at least about 25%, at least about 50%, at least about 75%, at leastabout 85%, or at least about 90%, up to total inhibition of growth ofthe tumor, when compared to a suitable control. Thus, in theseembodiments, “effective amounts” of an ABP are amounts that aresufficient to reduce tumor growth rate by at least about 5%, at leastabout 10%, at least about 20%, at least about 25%, at least about 50%,at least about 75%, at least about 85%, or at least about 90%, up tototal inhibition of tumor growth, when compared to a suitable control.In an experimental animal system, a suitable control may be tumor growthrate in a genetically identical animal not treated with the agent. Innon-experimental systems, a suitable control may be the tumor load ortumor growth rate present before administering the agent. Other suitablecontrols may be a placebo control.

Whether growth of a tumor is inhibited can be determined using any knownmethod, including, but not limited to, an in vivo assay for tumorgrowth; an in vitro proliferation assay; a ³H-thymidine uptake assay;and the like.

Non-Therapeutic Methods of Using ABPs

ABPs can also be used in diagnostic and/or imaging methods. For example,ABPs having an enzymatically cleavable CM can be used to detect thepresence or absence of an enzyme that is capable of cleaving the CM.Such ABPs can be used in diagnostics, which can include in vivodetection (e.g., qualitative or quantitative) of enzyme activity (or, insome embodiments, an environment of increased reduction potential suchas that which can provide for reduction of a disulfide bond) accompaniedby presence of a target of interest through measured accumulation ofactivated ABPs in a given tissue of a given host organism.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The TBM can be onethat binds a target antigen. Using methods familiar to one skilled inthe art, a detectable label (e.g., a fluorescent label) can beconjugated to a TBM or other region of an ABP. Suitable detectablelabels are discussed in the context of the above screening methods andadditional specific examples are provided below. Using a TBM specific toa protein or peptide of the disease state, along with a protease whoseactivity is elevated in the disease tissue of interest, ABPs willexhibit increased rate of binding to disease tissue relative to tissueswhere the CM specific enzyme is not present at a detectable level or ispresent at a lower level than in disease tissue. Since small proteinsand peptides are rapidly cleared from the blood by the renal filtrationsystem, and because the enzyme specific for the CM is not present at adetectable level (or is present at lower levels in non-diseasedtissues), accumulation of activated ABP in the diseased tissue isenhanced relative to non-disease tissues.

In another example, ABPs can be used in to detect the presence orabsence of a cleaving agent in a sample. For example, where the ABPcontains a CM susceptible to cleavage by an enzyme, the ABP can be usedto detect (either qualitatively or quantitatively) the presence of anenzyme in the sample. In another example, where the ABP contains a CMsusceptible to cleavage by reducing agent, the ABP can be used to detect(either qualitatively or quantitatively) the presence of reducingconditions in a sample. To facilitate analysis in these methods, the ABPcan be detectably labeled, and can be bound to a support (e.g., a solidsupport, such as a slide or bead). The detectable label can bepositioned on a portion of the ABP that is released following cleavage.The assay can be conducted by, for example, contacting the immobilized,detectably labeled ABP with a sample suspected of containing an enzymeand/or reducing agent for a time sufficient for cleavage to occur, thenwashing to remove excess sample and contaminants. The presence orabsence of the cleaving agent (e.g., enzyme or reducing agent) in thesample is then assessed by a change in detectable signal of the ABPprior to contacting with the sample (e.g., a reduction in detectablesignal due to cleavage of the ABP by the cleaving agent in the sampleand the removal of an ABP fragment to which the detectable label isattached as a result of such cleavage.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the TBMof the ABP. Thus, the in vitro assays can be adapted to assess thepresence or absence of a cleaving agent and the presence or absence of atarget of interest. The presence or absence of the cleaving agent can bedetected by a decrease in detectable label of the ABP as describedabove, and the presence or absence of the target can be detected bydetection of a target-TBM complex, e.g., by use of a detectably labeledanti-target antibody.

As discussed above, the ABPs disclosed herein can comprise a detectablelabel. In one embodiment, the ABP comprises a detectable label which canbe used as a diagnostic agent. Non-limiting examples of detectablelabels that can be used as diagnostic agents include imaging agentscontaining radioisotopes such as indium or technetium; contrastingagents for MRI and other applications containing iodine, gadolinium oriron oxide; enzymes such as horse radish peroxidase, alkalinephosphatase, or β-galactosidase; fluorescent substances and fluorophoressuch as GFP, europium derivatives; luminescent substances such asN-methylacrydium derivatives or the like.

The rupture of vulnerable plaque and the subsequent formation of a bloodclot are believed to cause the vast majority of heart attacks. Effectivetargeting of vulnerable plaques can enable the delivery of stabilizingtherapeutics to reduce the likelihood of rupture.

VCAM-1 is upregulated both in regions prone to atherosclerosis as wellas at the borders of established lesions. Iiyama, et al. (1999)Circulation Research, Am Heart Assoc. 85: 199-207. Collagenases, such asMMP-1, MMP-8 and MMP-13, are overexpressed in human atheroma which maycontribute to the rupture of atheromatous plaques. Fricker, J. (2002)Drug Discov Today 7(2): 86-88.

In one example, ABPs disclosed herein find use in diagnostic and/orimaging methods designed to detect and/or label atherosclerotic plaques,e.g., vulnerable atherosclerotic plaques. By targeting proteinsassociated with atherosclerotic plaques, ABPs can be used to detectand/or label such plaques. For example, ABPs comprising an anti-VCAM-1ABD and a detectable label find use in methods designed to detect and/orlabel atherosclerotic plaques. These ABPs can be tested in animalmodels, such as ApoE mice.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Methods and Materials

The following methods and materials were used in Examples 1-5 below.

Cloning and expression experiments were performed using E. coli strainMC1061. Cells were grown overnight at 37° C. in LB medium andchoramphenicol. Cultures were then diluted 1:50 into LB mediumcontaining chloramphenicol, grown for 2 hours at 37° C., and substrateexpression was induced with 0.2% L-(+)-arabinose at 37° C. for 2 hours.Approximately 2×10⁸ cells were centrifuged at 5000 rpm for 5 minutes,washed once with 50 mM Tris-Cl (pH 7.5) supplemented with 20 mM NaCl/2mM CaCl₂/100 μM ZnCl₂, and resuspended in 10 uL of Tris-Cl buffer.

In experiments involving addition of enzyme, 30 nM MMP-2 was included inthe Tris-Cl buffer (no enzyme added to the control reaction), and thereaction mixture was incubated at room temperature for 2 hours. Cellswere then removed and diluted 1:100 in PBS (pH 7.4) to stop thereaction, pelleted by centrifugation, and resuspended in 30 microlitersof refrigerated PBS containing 25 nM biotinylated VEGF. After incubationin the refrigerator on a rotary shaker for 45 minutes, cells werepelleted at 4° C. and resuspended in refrigerated PBS containing 50 nMStreptavidin-Phycoerythrin fluorescent conjugate. After incubation inthe refrigerator on a rotary shaker for 45 minutes, cells were pelletedat 4° C. and resuspended in PBS and red fluorescence was measured foranalysis or sorting on a FACSAria cell sorter. The fluorescence of cellstreated with enzyme was compared to control samples to determine theincrease in VEGF binding.

All switch constructs and libraries were displayed on the surface of E.coli bacteria using the N-terminus of the circularly permuted outermembrane protein X (CPX).

It should be noted that in describing the clones identified in theexperiments below, the following naming conventions are usedinterchangeably: #-#X-# and #.#X.#, where # is a numerical identifierand X is a alphabetical identifier.

Example 1 Construction of Polypeptides Having a VEGF Binding Moiety anda Moiety Cleavable by an Enzyme

As discussed above, the activatable binding polypeptides (ABPs) includea target binding moiety (TBM) and a cleavable moiety (CM), where the CMcontains a substrate for a protease. As a first step in the productionof ABPs, constructs for display on a bacterial cell surface weregenerated containing a VEGF binding sequence (to act as the TBM) and anamino acid sequence that is cleavable by matrix metalloprotease-2(MMP-2). The amino acid sequence of T7 antigen was included at theN-terminus as an immunodetectable tag to facilitate detection ofuncleaved product. Specifically binding of a detectably labeled anti-T7antibody was indicative of uncleaved ABP. The amino acid sequence ofthis construct without the cell surface anchoring sequences is providedbelow as SEQ ID NO: 12. FIG. 3 provides a schematic of the construct inthe presence and absence of enzyme (see upper right panel for details).

TABLE 3 SEQ ID NO: 12: N-Terminus T 7 Substrate VEGF Binder GQSGQMASMTGGQQM GGSG PLGLAG GGSG NFGYGKWEWDYGKWLEKVG

The ability of the construct to bind labeled VEGF in the presence orabsence of MMP-2 was tested. Bacteria displaying the construct on itssurface were incubated in the presence of labeled VEGF either in thepresence or absence of MMP-2 (FIG. 3, left panels). Cleavage of theconstruct by the protease was confirmed by incubating the cells eitherin the presence or absence of a detectably-labeled anti-T7 antigenantibody (FIG. 3, right panels). Binding of either labeled VEGF orlabeled anti-T7 antibody was assessed by FACS. As shown in FIG. 3, whenthe construct is contacted with labeled VEGF in either the presence orabsence of MMP-2, the labeled VEGF is able to bind the VEGF bindersequence, indicating that the presence of the enzyme substrate does notsubstantially interfere with VEGF binding to the TBM of the construct.FIG. 3 also confirms that MMP-2 cleaved the PLGLAG substrate of theconstruct, as indicated by an approximately 16.5 fold decrease inaverage fluorescence of the construct in the presence of the enzyme.

These data illustrate that VEGF binding is not substantially impaired bythe presence of the MMP-2 substrate and that the MMP-2 substrateutilized in the T7 control polypeptide is a candidate enzyme substratefor use as a CM in an ABP.

Example 2 ABP Having a Cysteine-Constrained Loop

One strategy for “masking” a target binding moiety (TBM) in an ABP is toprovide the ABP in a “loop” that sterically hinders access of target tothe TBM. In this strategy, cysteines are positioned at or near theN-terminus and C-terminus of the ABP, such that upon formation of adisulfide bond between the cysteines, the TBM is masked.

An exemplary ABP is illustrated in FIG. 4. This ABP includes acysteine-containing flexible linker sequence positioned N-terminal of aMMP-2 substrate (the cleavable moiety (CM), indicated as “substrate”below), which in turn was N-terminal of a VEGF binder as the TBM. Aflexible linker was positioned between the CM and TBM. The sequence isprovided below as SEQ ID NO: 13.

TABLE 4 SEQ ID NO: 13: N-Terminus Cysteine Substrate Linker VEGF BinderGQSGQ GCGSG PLGLAG GGSG NFGYGKWEWDYGKWLEKVGGC

A control (“GControl”) that lacked the cysteine-cysteine disulfide bondwas also constructed. The sequence of the GS control is provided belowin SEQ ID NO: 14.

TABLE 5 SEQ ID NO: 14: N- Sub- Terminus Insert strate VEGF Binder GQSGQ(GGS)₅ PLGLAG GGSG NFGYGKWEWDYGKWLEKVGGG

These constructs were then tested for the ability to bind labeled VEGFin the presence or in the absence of MMP-2 as described above.

The ABP of FIG. 4 was displayed on the surface of a bacterial cell andcontacted with labeled VEGF in presence and absence of MMP-2 enzyme.FACS analysis to detect VEGF-labeled cells was performed to determinewhether the ABP demonstrated switching behavior as compared to thecontrol polypeptide lacking the cysteine-cysteine disulfide bond. Asillustrated in FIG. 5, binding of labeled VEGF was increased in thepresence of enzyme compared to in the absence of enzyme, as evidenced byan approximately 2.6 fold increase in fluorescence after MMP-2 treatment(FIG. 5, right panels). A similar increase in VEGF binding was not seenin the GS control polypeptide.

These data illustrate that cleavage of the substrate by MMP-2 providedfor enhanced binding of VEGF to the ABP as compared to the binding ofVEGF to the ABP in the absence of MMP-2. In addition, the cysteineloop-containing ABP exhibited a more enhanced “switchable” VEGF-bindingphenotype as compared to the GS control. The level of VEGF binding tocleaved cysteine loop-containing ABP relative to uncleaved cysteineloop-containing ABP was greater than the level of VEGF binding tocleaved GS control relative to uncleaved GS control.

Example 3 Screening of ABP Libraries

In order to identify further ABPs having desired “switching”characteristics (i.e., decreased target binding when in an uncleavedconformation relative to target binding when in a cleaved conformation),libraries of candidate ABPs having different variable amino acidsequences in the masking moieties (MMs) and varying positions of thecysteine in the MM were generated. The amino acid sequences of exemplarylibraries are provided below as SEQ ID NOs. 15-18 in Table 6. “X”represents a randomized amino acid sequence. Glycine was included inorder to impart flexibility to the MM.

TABLE 6 N-Terminus Library Substrate Linker VEGF Binder 1 (SEQ ID GQSGQCX₆G PLGLAG GGSG NFGYGKWEWDYGKWLEKVGGC NO: 15 2 (SEQ ID GQSGQ X₅G PLGLAGGGSG NFGYGKWEWDYGKWLEKVGGC NO: 16) 3 (SEQ ID GQSGQ XCX₃ PLGLAG GGSGNFGYGKWEWDYGKWLEKVGGC NO: 17) 4 (SEQ ID GQSGQ X₅G PLGLAG GGSGNFGYGKWEWDYGKWLEKVGGG NO: 18) X = any amino acid

FIG. 6 provides a schematic of the library constructs, and illustratesthe construct design to provide cysteines of the construct (underlinedresidues) which can form a disulfide bridge, thereby constraining theconformation of the construct in the uncleaved state.

FIG. 7 provides a schematic illustrating the screening/sorting methodused to identify candidates that display the switchable phenotype. Thelibraries were introduced via expression vectors resulting in display ofthe ABP polypeptides on the surface of the bacterial cells. Theresulting display library contained more than 3×10⁸ transformants. Afterexpansion of the libraries by culture (“Grow Library”), cells displayingthe ABP polypeptides were then treated with MMP-2 enzyme to provide forcleavage of the cleavable substrate moiety. MMP-2 treated cells werethen contacted with fluorescently labeled VEGF and the cells were sortedby FACS to isolate cells displaying ABPs which bound VEGF after cleavagewith MMP-2. The cells that displayed VEGF-binding constructs were thenexpanded by growth in culture (“Grow Library”). The cells were thencontacted with labeled VEGF and sorted by FACS to isolate cellsdisplaying ABPs which failed to bind labeled VEGF in the absence ofMMP-2. These steps represented one “cycle” of the screening procedure.The cells can then be subjected to additional cycles by expansion bygrowth in culture (“Grow Library”), and again subjecting the culture toall or part of the screening steps.

It should be noted that library screening and sorting could also beinitiated by first selecting for candidates that do not bind labeledVEGF in the absence of enzyme treatment (i.e., do not bind VEGF when notcleaved).

Exemplary data for one of the libraries is provided in FIG. 8. After 1.5cycles of selection (i.e., one complete cycle of enzyme treatment,sorting, VEGF-binding, sorting; followed by a half-cycle of enzymetreatment and sorting), libraries exhibited a marked improvement in the“switchable” phenotype, with binding of labeled VEGF in the absence ofenzyme (FIG. 8, top right panel) being significantly less than in thepresence of enzyme (FIG. 8, bottom right panel). In addition, asillustrated in FIG. 8, left panels, the unsorted library exhibited aless significant switchable phenotype, confirming that theselection/sorting method is effective in enrichment of the library forcells displaying an ABP having a desired switchable phenotype.

In addition, selected clones demonstrated improved “switching” activitycompared to cysteine-constrained controls. FIG. 9 shows foldfluorescence increase after cleavage with MMP-2 for selected libraryclones identified from each of libraries 1-4. The selected clones ofFIG. 9, identified after screening the libraries identified in FIG. 6,showed a modest improvement when compared with clones derived from ascreen of a random library. For example, of the 40 clones identified inFIG. 9, six demonstrated a 3-fold increase in fluorescence. The averagefluorescence increase for the 40 clones was approximately 2 fold. Forclones derived from the random library, 2 of 23 clones demonstrated a3-fold increase in fluorescence. The average fluorescence increase forthe random library of clones was approximately 1.5 fold.

The amino acid sequences of clones exhibiting the most marked“switching” phenotype (also referred to as an enzymatically“activatable” phenotype) are provided in FIG. 10.

In a further screen, several additional clones were identified whichexhibited a marked “switching” phenotype. The fold fluorescence increaseafter enzyme treatment for these clones is shown in FIG. 24.

FIG. 25 shows switching activity for selected clones based on foldfluorescence increase after enzyme cleavage. Each of clones 4-2A-11(GEDEEG: SEQ ID NO: 19 including fixed G residue), 2-2A-5 (PEWGCG: SEQID NO: 11 including fixed G residue), 2-3B-5 (CEYAFG: SEQ ID NO: 20including fixed G residue), 1-3A-4 (CSMYWMRG: SEQ ID NO: 21 includingfixed C and G residues), and 2-3B-6 (EYEGEG: SEQ ID NO: 22 includingfixed G residue) exhibit improved switching activity relative tocontrol, with clone 2-3B-5 exhibiting an approximate 10-fold increase influorescence relative to an increase in fluorescence of less thanapproximately 2-fold for the GS control.

Example 4 The Switchable Phenotype is Attributable to Cleavage of theCleavable Moiety

In general, the switchable phenotype is due to a change in conformationof the ABP that allows for more or less access of the target to thetarget binding moiety (TBM). Where the ABP contains cysteines capable offorming a disulfide bridge, the switchable phenotype could be a resultof at least two different mechanisms: 1) cleavage of the ABP at theenzyme cleavage site; or 2) reduction of the disulfide bond betweencysteines positioned near the N- and C-termini of the ABP.

For example, switching behavior was observed in ABPs that contained a MMthat lacked a cysteine. Three clones, each with a different 5 amino acidMM library sequence, were tested for the ability to bind labeled VEGF inthe presence and absence of MMP-2. As seen in FIG. 11, clones with MMlibrary amino acid sequences of GEDEE: SEQ ID NO: 23 (GEDEEG: SEQ ID NO:19 including fixed G residue) and DDMEE: SEQ ID NO: 24 (DDMEEG: SEQ IDNO: 25 including fixed G residue) showed improved binding in thepresence of MMP-2 despite the lack of cysteine residues in the MM. Thisresult indicates that the disulfide bond linkage is not necessarilyrequired in order for an ABP to demonstrate the desired switchingactivity.

Additional cysteine and non-cysteine containing MM sequences identifiedaccording to the screening methods described herein are shown in FIG.22.

As indicated above, the switchable phenotype could potentially resultfrom the disruption of the disulfide bond linkage between a cysteineresidue in the MM and a cysteine residue adjacent the TBM. Thispossibility was verified by testing clones in the presence and absenceof disulfide bond reducing conditions. As indicated in FIG. 12, clone2.2A.5 (a clone that was the product of the screening procedure above)and a cysteine constrained parent (i.e., a design or “trial” sequencethat shows 2-fold switching activity, sequence given in FIG. 4) that wasnot the product of the screening procedure were each tested for theability to bind labeled VEGF in the presence and absence DTT reducingconditions. Both the cysteine constrained parent and clone 2.2A.5 showedincreased binding of labeled VEGF after reduction of the disulfide bondwith DTT.

However, the screening procedure of the Example above provides for anenhanced switching phenotype over that associated with conformationchange as a result of reduction of disulfide bonds. This is evidenced bythe results of analysis of the switching phenotype of thecysteine-constrained parent construct and clone 2.2A.5. The K_(d,app)values for VEGF binding by the cysteine constrained parent weredetermined in the presence and absence of MMP-2 and compared withK_(d,app) values for VEGF binding by the library clone 2.2A.5 in thepresence and absence of MMP-2. Clone 2.2A.5 showed an approximately 4.8fold improvement in K_(d) in the presence of enzyme (FIG. 14) ascompared with an approximately 3.4 fold improvement in K_(d) in thepresence of enzyme for the cysteine constrained parent (FIG. 13).

In an additional experiment, the fluorescence values for a cysteineconstrained loop structure were determined in the presence and absenceof MMP-2 and compared with the fluorescence values for library clones4.2A.11 (a.k.a. 4-2A-11) and 2.3B.5 (a.k.a. 2-3B-5) in the presence andabsence of MMP-2. Clone 4.2A.11 showed an approximate 5.7 fold increasein fluorescence in the presence of enzyme (FIG. 23) and clone 2.3B.5showed an approximate 11.8 fold increase in fluorescence in the presenceof enzyme as compared with an approximately 3 fold increase influorescence in the presence of enzyme for the cysteine constrained loopstructure (FIG. 23).

These results indicate that optimization of a cysteine constrained ABPto provide for an enhanced switchable phenotype can be achieved byscreening an ABP library containing MMs with variable amino acidsequences.

Example 5 Screening for Desired Dynamic Range

Dynamic range can be enhanced by at least two mechanisms; 1) the switchoff state can be improved by improving the MM to prevent binding betweenthe target and TBM, and 2) the affinity of the TBM can be improved aftersubstrate cleavage that results in the MM motif acting as cooperativetarget binding element (FIGS. 4 and 5). Screening for expanded dynamicrange can, in certain embodiments, be effectively accomplished by usingalternating separations, (e.g. using FACS) that use differentconcentrations of the target protein for the “A” and “B” stepsrepresented in FIG. 2. To identify in separation “A” binders that mayhave improved affinity relative to the TBM when used alone (i.e. outsideof the switch context), a target ceontration of 10 nM was used that isapproximately 10-fold below the expected dissociation constant (100 nM)of the TBM. Cells exhibiting the highest level of fluorescence were thencollected using FACS, and amplified by overnight growth. Then, toimprove the off-state (i.e. ability to bind the target in the absence ofthe protease), the cell population was incubated with 1 μM VEGF (aconcentration significantly above the KD of the TBM), and cellsexhibiting low levels of fluorescence were collected. This processresulted in a pool of ABPs with a greater dynamic range, than a processusing the same concentration of the target in both A and B sorts. Anadditional embodiment of a screening method using different targetconcentrations for steps “A” and “B” is depicted in FIG. 21, wherein a25 nM VEGF concentration is used for the “A” sort and a 250 nM VEGFconcentration is used for the “B” sort. The enriched cell populationsresulting from selection using FACS are shown in FIGS. 15 and 16 forsorts 1A, 1B, 2A, 2B, and 3A where A and B are positive and negativeselections, respectively. Enrichment of the library for ABPs can bedetermined by comparing the unsorted library populations' fluorescencedistribution change from protease treatment, to that of the round 3Apopulation before and after protease treatment. The later enriched poolshows an average dynamic range of approximately four-fold as indicatedby the four fold increase in cell fluorescence after enzyme treatment(FIG. 16, Right-hand panel “Sort 3A 25 nM VEGF”).

Example 6 Soluble Protein Fusions Demonstrate Enzyme Mediated Binding

In order to demonstrate the activity of ABPs in soluble form, C-terminalmaltose-binding protein (MBP) fusions of VEGF binding clones and VEGFbinding ABPs were constructed and tested.

Methods and Materials

VEGF was immobilized to the Biacore™ CM5 sensor chip surface using thestandard amine coupling kit. An NHS/EDC mixture was injected first toactivate the surface using the surface preparation wizard in theBiacore™ software. Then, 25 ug/mL concentration of VEGF was injecteduntil the desired immobilization amount was reached (typically ˜5000response units). The surface is then blocked with ethanolamine. Acontrol reaction was performed on another surface with NHS/EDC thenEthanolamine.

After the VEGF is covalently immobilized, the maltose-binding protein(MBP) fusions of VEGF binding or VEGF binding ABP clones were injectedover both the VEGF surface and the control surface. Injections weretypically for 1 minute, with a few minutes of dissociation time aftereach injection. The clones were injected both with and without 30 nMMMP-2 enzyme. For analysis, the signal on the VEGF surface minus thesignal on the control surface is the binding response (in RU). Cloneswere compared in triplicate, with and without enzyme, at cloneconcentrations of up to 15 micromolar.

Results

As indicated in FIG. 27, exemplary MBP-ABP fusions retained their enzymemediated VEGF binding properties, with the 2.3B.5 (2-3B-5) fusionexhibiting an approximate 4-fold increase in binding response in thepresence of MMP-2 enzyme. A similar increase in binding response in thepresence of enzyme was not seen for the VEGF binding peptide controls.These results demonstrate retained “switching activity” for solubleABPs.

Example 7 Identification of Peptide Sequences for Use as MMs in anAnti-VCAM-1 ABP

The following materials and methods were utilized to identify peptidesequences for use as masking moieties (MM) in ABPs wherein the targetbinding moiety (TBM) comprises an antigen binding domain (ABD) of ananti-VCAM-1 scFv.

Methods and Materials

Magnetic-Activated Cell Sorting (MACS) (one round) and FluorescenceActivated Cell Sorting (FACS) (three rounds) were utilized to enrich forclones exhibiting strong binding to anti-VCAM-1 scFv.

Bacterial cells displaying selected peptide sequences were sorted byFACS after contacting with 1 nM anti-biotin phycoerythin (PE) or 50 nMbiotinylated anti-VCAM scFv followed by 1 nM anti-biotin PE.

Results

The following peptide sequences were identified as a result of thereferenced MACS and FACS analysis:

TABLE 7 Clone Amino Acid Sequence BBB-08 GVVLTTMNFWDWITV (SEQ ID NO: 26)BBB-09 WADWARSWEAIVGMA (SEQ ID NO: 27) BBB-16RGMDMYWAEIIYGAA (SEQ ID NO: 28)

As demonstrated by FACS analysis in FIG. 28, each of clones BBB-08,BBB-09 and BBB-16 showed significant binding to anti-VCAM-1 scFV. Of thethree clones, BBB-08 showed the highest level of binding to anti-VCAM-1scFV with an average fluorescence value of 2,625 following incubationwith anti-VCAM-1 scFV as compared to an average fluorescence value of142 in the absence of anti-VCAM-1 scFV. As such, these peptides arelikely candidates for functional MMs in ABPs comprising an antigenbinding domain (ABD) of an anti-VCAM-1 scFv as a TBM.

Example 8 Prophetic ABPs Comprising an Anti-VCAM-1 Antigen BindingDomain

Prophetic examples of ABPs comprising an anti-VCAM-1 scFv (MK271) aredescribed herein. These ABPS will be inactive under normal conditionsdue to an attached MM. When the scFv reaches the site of plaque,however, matrix metalloproteinase-1 will cleave a substrate linkerconnecting the peptide inhibitor to the scFv allowing it to bind toVCAM-1. A representation of this process is set forth in FIG. 35.Bacterial cell surface display was used as described in Example 7 tofind suitable MMs for the antibody. In the prophetic ABPs, selected MMsare combined with an enzyme substrate to be used as a trigger to createa scFv construct that becomes competent for targeted binding afterprotease activation.

Prophetic ABPs utilizing the peptide sequences identified in Example 7and comprising an antigen binding domain (ABD) of an anti-VCAM-1 scFvare provided in FIGS. 29, 30 and 31. These prophetic ABP sequencescomprises an OmpA periplasmic signal sequence, a Flag tag, a His tag, apeptide sequence which binds anti-VCAM scFV, an MMP-1 substrate and ananti-VCAM scFV sequence. Lower-case, non-bold letters indicate linkersequences. The sequence of the anti-VCAM scFV is indicated byunderlining and all caps. The MM sequence is indicated in bold and allcaps.

Construction of Protease Activated Antibody

Genes encoding ABPs comprising a VCAM-1 antibody in single-chain formare produced by overlap extension PCR or total gene synthesis withflanking SfiI restriction enzyme sites, digested with SfiI and ligatedinto a similarly digested expression plasmid pBAD33, or any othersuitable bacterial, yeast, or mammalian expression vector familiar toone skilled in the art. Full length antibodies could alternatively beproduced using commercially available expression vectors incorporatinghalf-life extending moieties (e.g. the Fc region of an IgG, serumalbumin, or transferrin) and methods familiar to one skilled in the art.The expression plasmid is then transformed or transfected into anappropriate expression host such as BL21 for E. coli or HEK293t cells.Single chain antibodies are harvested from overnight cultures using aPeriplasmic fraction extraction kit (Pierce), and purified byimmobilized metal ion affinity chromatography, and by size exclusionchromatography.

In some instances, it may be desirable to produce ABPs comprisingantibodies as insoluble aggregates in the cytoplasm. In such cases, thesignal sequence can be removed, and an appropriate affinity tag (6×His)can be introduced at the C-terminus to aid purification. Prophetic ABPsequences for cytoplasmic expression (as inclusion bodies) are providedin FIGS. 32, 33 and 34. Lower-case, non-bold letters indicate linkersequences. The sequence of the anti-VCAM scFV is indicated byunderlining and all caps. The MM sequence is indicated in bold and allcaps.

Assay for Antibody Switching Activity In Vitro

Aliquots of protease-activated antibodies, at a concentration of 1 pM-1μM) are incubated in a buffered aqueous solution separately with 0 and50 nM MMP-1 for 3 hrs. The reaction mixtures are then assayed forbinding using ELISA or surface Plasmon resonance with immobilizedantigen VCAM-1. An increase in binding activity for the ABP afterprotease treatment is indicated by an increase in resonance units whenusing BIAcore™ SPR instrumentation. The change in apparent dissociationconstant (Kdiss) as a result of MMP cleavage can then be calculatedaccording the instrument manufacturer's instructions (BIAcore, GEHealthcare).

Example 9 Cloning of the Anti-VEGF scFV TBM

In certain embodiments, the ABP includes a TBM that contains an ABD, aMM, and a CM. In this and following examples an ABP containing a maskedMMP-9 cleavable anti-VEGF scFv (target=VEGF; TBM=anti-VEGF single chainFv) was constructed. As a first step in the production of such an ABP,constructs containing an anti-VEGF scFv were generated (the TBM). Ananti-VEGF scFv TBM (V_(L)-linker L-V_(H)) was designed from thepublished sequence of ranibizumab (Genetech, Chen, Y., Wiesmann, C.,Fuh, G., Li, B., Christinger, H., McKay, P., de Vos, A. M., Lowman, H.B. (1999) Selection and Analysis of an Optimized Anti-VEGF Antibody:Crystal Structure of an Affinity-matured Fab in Complex with Antigen J.Mol. Biol. 293, 865-881) and synthesized by Codon Devices (Cambridge,Mass.).

Ranibizumab is a monoclonal antibody Fab fragment derived from the sameparent murine antibody as bevacizumab (Presta L G, Chen H, O'Connor S J,et al Humanization of an anti-vascular endothelial growth factormonoclonal antibody for the therapy of solid tumors and other disorders.Cancer Res, 57: 4593-9, 1997). It is much smaller than the parentmolecule and has been affinity matured to provide stronger binding toVEGF-A. Ranibizumab binds to and inhibits all subtypes of vascularendothelial growth factor A (VEGF-A). A His6 tag at the N-terminus and aTEV protease cleavage site were included in the design. The TEV proteaseis a protease isolated from tobacco etch virus, is very specific, and isused to separate fusion proteins following purification. The anti-VEGFscFv nucleotide and amino acid sequences are provided below in SEQ IDNOs: 29 and 30 respectively.

TABLE 8 SEQ ID NO: 29: anti-VEGF scFv TBM nucleotide sequencegatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcg

TABLE 9 SEQ ID NO: 30: anti-VEGF scFv TBM amino acid sequenceDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVS

Example 10 Screening and Identification of MMs for Anti-VEGF scfv

Ranibizumab was used to screen a pooled random peptide library,consisting of peptides that are X₁₅ (8.3×10⁹), X₄CX₇CX₄ (3.6×10⁹), orX₁₂CX₃ (1.1×10⁹), where X is any amino acid and the number representsthe total diversity of the library. The total diversity of the pooledlibrary was 1.3×10¹⁰. The screening consisted of one round of MACS andtwo rounds of FACS sorting. In the first round MACS screen, 1×10¹¹ cellswere probed with 150 nM biotinylated-ranibizumab, and 5.5×10⁷ bindingcells were isolated. In the first FACS screen, positive cells isolatedin the MACS screen were probed with 500 nM biotinylated-ranibizumab, andvisualized with neutrAvidin-PE (Molecular Probes, Eugene, Oreg.). Thesecond and third rounds of FACS selections were done with 500 nM andthen 100 nM Alexa-labeled ranibizumab in the presence of 20 uM IgG.Individual clones were sequenced and subsequently verified for theirability to bind anti-VEGF scfv by FACS analysis. Amino acid sequences ofMMs for anti-VEGF scFv are provided in Table 10 below. (These sequenceswill hereafter be referred to as 283MM, 292MM, 306MM, etc.)

TABLE 10 MMs for anti-VEGF scFv JS283 ATAVWNSMVKQSCYMQG (SEQ ID NO: 31)JS292 GHGMCYTILEDHCDRVR (SEQ ID NO: 32) JS306 PCSEWQSMVQPRCYYGG(SEQ ID NO: 33) JS308 NVECCQNYNLWNCCGGR (SEQ ID NO: 34) JS311VHAWEQLVIQELYHC (SEQ ID NO: 35) JS313 GVGLCYTILEQWCEMGR (SEQ ID NO: 36)JS314 RPPCCRDYSILECCKSD (SEQ ID NO: 37) JS315 GAMACYNIFEYWCSAMK(SEQ ID NO: 38)

Example 10 Construction of the ABP: MMP-9 Cleavable, Masked-Anti-VEGFscFV Vectors

A CM (substrate for MMP-9) was fused to the masked anti-VEGF scFvconstruct to provide a cleavable, masked ABP. An exemplary construct isprovided in FIG. 38. Several exemplary ABP constructs and sequencescontaining different CMs are described in great detail below. Primersutilized for construction of the exemplary constructs are represented inTable 11 below.

TABLE 11 Primers utilized for construction of MMP-9 cleavable,masked- anti-VEGF scFv CX0233 5′gaattcatgggccatcaccatcaccatcacggtgggg3′(SEQ ID NO: 39) CX0249 5′gtgagtaagcttttattacgacactgtaaccagagtaccctgg3′(SEQ ID NO: 40) CX02705′gtggcatgtgcacttggccaccttggcccactcgagctggccagactggccctgaaaatacagattttccc3′(SEQ ID NO: 41) CX02715′gagtgggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcc3′(SEQ ID NO: 42) CX0288 5′ ttcgagctcgaacaacaacaacaataacaataacaacaac3′(SEQ ID NO: 43) CX0289 5′ gctttcaccgcaggtacttccgtagctggccagtctggcc3′(SEQ ID NO: 44) CX0290 5′ cgctccatgggccaccttggccgctgccaccagaaccgcc3′(SEQ ID NO: 45) CX0308 5′ gcccagccggccatggccggccagtctggccagctcgagt3′(SEQ ID NO: 46) CX0310 5′ccagtgccaagcttttagtggtgatggtgatgatgcgacactgtaaccagagtaccctggcc3′(SEQ ID NO: 47) CX0312 5′cttgtcacgaattcgggccagtctggccagctcgagt3′(SEQ ID NO: 48) CXO3145′cagatctaaccatggcgccgctaccgcccgacactgtaaccagagtaccctg3′ (SEQ ID NO: 49)

Cloning and Expression of the ABP: a MMP-9 Cleavable, Masked Anti-VEGFscFv as a MBP Fusion

Cloning: A MBP:anti-VEGF scFv TBM fusion was cloned. The MBP (maltosebinding protein) expresses well in E. coli, as a fusion protein, and canbe purified on a maltose column, a method well known in the art to makefusion proteins. In this example, the MBP was used to separate themasked scFv. The His6 tagged Anti-VEGF scFv TBM was cloned into thepMal-c4x vector (NEB) as a C-terminal fusion with MBP using the EcoRIand HindIII restriction sites in the multiple cloning site (MCS). Theprimers CX0233 and CX0249 (Table 11) were used to amplify the Anti-VEGFscFv TBM and introduce the EcoRI and HindIII sites, respectively.

The accepting vector for the ABP (the peptide MM, the anti-VEGF scFv TBMand the MMP-9 CM) was synthesized using polymerase chain reaction (PCR)with the overlapping primers CX0271 and CX0270 which placed the cloningsite for the peptide MM's, linker sequences, and MMP-9 CM protease sitebetween the TEV protease site and the anti-VEGF scFv TBM. The primersCX0271 and CX0249 (Table 11) were used to amplify the C-terminal portionof the construct, while the primers CX0270 and CX0288 (Table 11) wereused to amplify the N-terminal portion. Products from both the abovereactions were combined for a final PCR reaction using the outsideprimers, CX0249 and CX0288 (Table 11), and cloned into the pMal vectoras an MBP fusion using the SacI and HindIII restriction sites (SEQ IDNO: 50)

TABLE 12 SEQ ID NO: 50: MBP/MM accepting site/MMP-9 CM/Anti-VEGF scFvTBM vector nucleotide sequenceatgggccatcaccatcaccatcacggtggggaaaatctgtattttcagggccagtctggccagctcgagtgggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcg

The 306MM and 314MM (Table 10) were amplified from the ecpX displayvector using the primers CX0289 and CX0290 (Table 11), and directionallycloned into the N-terminally masked vector using the SfiI restrictionsites. The corresponding nucleotide and amino acid sequences areprovided in Table 13 below.

TABLE 13 SEQ ID NO: 51: MBP/306 MM/MMP-9 CM/Anti-VEGF scFv TBMnucleotide sequenceatgggccatcaccatcaccatcacggtggggaaaatctgtattttcagggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgSEQ ID NO: 52: MBP/306 MM/MMP-9 CM/Anti-VEGF scFv TBMamino acid sequenceMGHHHHHHGGENLYFQGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQG TLVTVSSEQ ID NO: 53: MBP/314 MM/MMP-9 CM/Anti-VEGF scFv TBMnucleotide sequenceatgggccatcaccatcaccatcacggtggggaaaatctgtattttcagggccagtctggccagcggccgccgtgttgccgtgattatagtattttggagtgctgtaagagtgatggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgSEQ ID NO: 54: MBP/314 MM/MMP-9 CM/Anti-VEGF scFv TBMamino acid sequenceMGHHHHHHGGENLYFQGQSGQRPPCCRDYSILECCKSDGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQ GTLVTVS

Expression:

Expression of the MBP:ABP fusions were conducted in a K12 TB1 strain ofE. coli. An ampicillin-resistant colony containing the desired constructwas used to inoculate a 5 ml overnight culture containing LB mediumsupplemented with 50 μg/mL Ampicillin. The entire overnight culture wasused to inoculate 500 mL of fresh LB medium supplemented with 50 μg/mLampicillin and 0.2% Glucose and allowed to grow at 37° C. shaking at 250rpm until an O.D. of 0.5 was reached. Isopropylthio-β-D-galactosidasewas then added to a final concentration of 0.3 mM and the culture wasallowed to grow for a further 3 hrs under the same conditions afterwhich the cells were harvested by centrifugation at 3000×g. Inclusionbodies were purified using standard methods. Briefly, 10 mls of BPER IIcell lysis reagent (Pierce). Insoluble material was collected bycentrifugation at 14,000×g and the soluble proteins were discarded. Theinsoluble materials were resuspended in 5 mls BPER II supplemented with1 mg/mL lysozyme and incubated on ice for 10 minutes after which 5 mlsof BPER II diluted in water 1:20 was added and the samples were spun at14,000×g. The supernatant was removed and the pellets were wash twice in1:20 BPER11. The purified inclusion bodies were solubilized in PBS 8 MUrea, 10 mM BME, pH 7.4.

The MBP fusion proteins were diluted to a concentration of approximately1 mg/mL and refolded using a stepwise dialysis in PBS pH 7.4 from 8 to 0M urea through 6, 4, 2, 0.5, and 0 M urea. At the 4, 2, and 0.5 M Ureasteps 0.2 M Arginine, 2 mM reduced Glutathione, and 0.5 mM oxidizedglutathione was added. The 0M Urea dialysis included 0.2 M Arginine.After removal of the urea, the proteins were dialyzed against 0.05 MArginine followed by and extensive dialysis against PBS pH 7.4. Alldialysis were conducted at 4° C. overnight. To remove aggregates, eachprotein was subjected to size exclusion chromatography on a sephacrylS-200 column. Fractions containing the correctly folded proteins wereconcentrated using an Amicon Ultra centrifugal filter.

Cloning and Expression of the ABP: a MMP-9 Cleavable, Masked Anti-VEGFscfv CHis Tag

Cloning:

The primers CX0308 and CX0310 (Table 11) were used to amplify and add aNcoI restriction site to the 5′ end and a HindIII restriction site andHis6 tag to the 3′ end, respectively, of the (MM accepting site/MMP-9CM/VEGFscFv TBM) vector which was subsequently cloned into a vectorcontaining the pelB signal peptide. Anti-VEGF scFv MMs were cloned aspreviously described. The corresponding nucleotide and amino acidsequences are provided in Table 14 below.

TABLE 14 SEQ ID NO: 55: 306 MM/MMP-9 CM/anti-VEGF scFv CHis TBMnucleotide sequenceggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgcatcatcaccatcaccacSEQ ID NO: 56: 306 MM/MMP-9 CM/anti-VEGF scFv CHis TBMamino acid sequenceGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSHHHHHHSEQ ID NO: 57: 314 MM/MMP-9 CM/anti-VEGF scFv CHis TBMnucleotide sequenceggccagtctggccagcggccgccgtgttgccgtgattatagtattttggagtgctgtaagagtgatggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgcatcatcaccatcaccactaaSEQ ID NO: 58: 314 MM/MMP-9 CM/anti-VEGF scFv CHis TBMamino acid sequenceGQSGQRPPCCRDYSILECCKSDGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSHHHHHH

Expression:

Expression of the Anti-VEGF scFv His ABPs was conducted in a K12 TB1strain of E. coli. An ampicillin-resistant colony containing the desiredconstruct was used to inoculate a 5 ml overnight culture containing LBmedium supplemented with 50 μg/mL Ampicillin. 2.5 ml of overnightculture was used to inoculate 250 mL of fresh LB medium supplementedwith 50 μg/mL ampicillin and 0.2% Glucose and allowed to grow at 37° C.shaking at 250 rpm until an O.D. of 1.0 was reached.Isopropylthio-β-D-galactosidas was then added to a final concentrationof 0.3 mM and the culture was allowed to grow for a further 5 hrs at 30°C. after which the cells were harvested by centrifugation at 3000×g. Theperiplasmic fraction was immediately purified using the lysozyme/osmoticshock method. Briefly, the cell pellet was resuspended in 3 mLs of 50 mMTris, 200 mM NaCl, 10 mM EDTA, 20% Sucrose, pH 7.4 and 2 uL/mL ready-uselysozyme solution was added. After a 15 min. incubation on ice, 1.5volumes of water (4.5 mLs) was added and the cells were incubated foranother 15 min. on ice. The soluble periplasmic fraction was recoveredby centrifugation at 14,000×g.

The Anti-VEGF scFv His proteins were partially purified using Ni-NTAresin. Crude periplasmic extracts were loaded onto 0.5 ml of Ni-NTAresin and washed with 50 mM phosphate, 300 mM NaCl, pH 7.4. His taggedproteins were eluted with 50 mM phosphate, 300 mM NaCl, 200 mMImidizale, pH 6.0. Proteins were concentrated to approximately 600 μLand buffer exchanged into PBS using Amicon Ultra centrifugalconcentrators.

Cloning and Expression of the ABP: a MMP-9 Cleavable, Masked Anti-VEGFscFv as Human Fc Fusion

Cloning:

The primers CX0312 and CX0314 (Table 11) were used to amplify thesequence encoding MMP-9 CM/Anti-VEGF scFv. The primers also includedsequences for a 5′ EcoRI restriction site and a 3′ NcoI restriction siteand linker sequence. Cutting the PCR amplified sequence with EcoRI andNcoI and subsequent cloning into the pFUSE-hIgG1-Fc2 vector generatedvectors for the expression of Fc fusion proteins. Anti-VEGF scFv TBM MMswere inserted into these vectors as previously described. Constructscontaining 306MM, 313MM, 314MM, 315MM, a non-binding MM (100MM), as wellas no MM were constructed and sequences verified. The correspondingnucleotide and amino acid sequences are provided below in Table 15.

TABLE 15 SEQ ID NO: 59: 306 MM/MMP-9 CM/anti-VEGF scfv-Fc TBMnucleotide sequenceggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgggcggtagcggcgccatggttagatctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaSEQ ID NO: 60: 306 MM/MMP-9 CM/anti-VEGF scFv-Fc TBM amino acid sequenceGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGKSEQ ID NO: 61: 314 MM/MMP-9 CM/anti-VEGF scfv-Fc TBM nucleotide sequenceggccagtctggccagcggccgccgtgttgccgtgattatagtattttggagtgctgtaagagtgatggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgggcggtagcggcgccatggttagatctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaSEQ ID NO: 62: 314 MM/MMP-9 CM/anti-VEGF scFv-Fc TBM amino acid sequenceGQSGQRPPCCRDYSILECCKSDGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK

Expression:

10 μg of expression vectors for 306 MM/MMP-9 CM/anti-VEGFscFv-Fc, 314MM/MMP-9 CM/anti-VEGFscFv-Fc or anti-VEGFscFv-Fc were introduced into10⁷ HEK-293 freestyle cells (Invitrogen, CA) by transfection usingtransfectamine 2000 as per manufacturer's protocol (Invitrogen, CA). Thetransfected cells were incubated for an additional 72 hours. Afterincubation, the conditioned media was harvested and cleared of cells anddebris by centrifugation. The conditioned media was assayed for activityby ELISA.

Example 11 Testing of a ABP

To measure the activation of the masked MMP-9 cleavable anti-VEGF ABPsby MMP-9, 100 μl of a 2 μg/ml PBS solution of VEGF was added tomicrowells (96 Well Easy Wash; Corning) and incubated overnight at 4° C.Wells were then blocked for 3×15 minute with 300 uL Superblock (Pierce).One hundred microliters of ABP (see below for details pertaining to eachconstruct), treated or untreated with MMP-9, were then added to wells inPBST, 10% Superblock and incubated at room temperature (RT) for 1 hr.All wash steps were done three times and performed with 300 ul PBST. Onehundred microliters of secondary detection reagent were then added andallowed to incubate at RT for 1 hr. Detection of HRP was completed using100 ul of TMB one (Pierce) solution. The reaction was stopped with 100μL of 1N HCL and the absorbance was measured at 450 nM.

ELISA Assay of ABP Construct Containing: MBP/MM/MMP-9 CM/Anti-VEGF scFvTBM

Two hundred microliters of biotinylated ABP in MMP-9 digestion buffer(50 mM Tris, 2 mM CaCl₂, 20 mM NaCl, 100 μM ZnCl₂, pH 6.8) at aconcentration of 200 nM was digested with 20U TEV protease overnight at4° C. to remove the MBP fusion partner. Samples were then incubated for3 hrs with or without ˜3U of MMP-9 at 37° C., diluted 1:1 to a finalconcentration of 100 nM in PBST, 10% Superblock, and added to the ELISAwells. Detection of the ABP was achieved with an Avidin-HRP conjugate ata dilution of 1:7500. MMP-9 activation of MMP-9 cleavable maskedMBP:anti-VEGF scFv ABP is presented in FIG. 39.

ELISA Assay of ABP Construct Containing: MM/MMP-9 CM/Anti-VEGF scFv His

Crude periplasmic extracts dialyzed in MMP-9 digestion buffer (150 μL)were incubated with or without ˜3U of MMP-9 for 3 hrs at 37° C. Sampleswere then diluted to 400 μL with PBST, 10% Superblock and added to theELISA wells. Detection of the ABP was achieved using an Anti-His6-HRPconjugate at a dilution of 1:5000. MMP-9 activation of MMP-9 cleavablemasked anti-VEGF scFv His ABP is presented in FIG. 40.

ELISA Assay of ABP Construct Containing: MM/MMP-9 CM/Anti-VEGF scFv-Fc

Fifty microliters of HEK cell supernatant was added to 200 μL MMP-9digestion buffer and incubated with or without ˜19U MMP-9 for 2 hrs at37° C. Samples were then diluted 1:1 in PBST, 10% Superblock and 100 μLwere added to the ELISA wells. Detection of the ABP was achieved usingAnti-human Fc-HRP conjugate at a dilution of 1:2500. MMP-9 activation ofMMP-9 cleavable masked anti-VEGF scFv-Fc is presented in FIG. 41.

Purification and Assay of ABP Construct Containing: MM/MMP-9CM/Anti-VEGF scFv-Fc

Anti-VEGF scFv Fc ABPs were purified using a Protein A columnchromatography. Briefly, 10 mLs of HEK cell supernatants were diluted1:1 with PBS and added to 0.5 mL Protein A resin pre-equilibrated inPBS. Columns were washed with 10 column volumes of PBS before elutingbound protein with 170 mM acetate, 300 mL NaCl pH. 2.5 and immediatelyneutralized 1 mL fractions with 200 μL of 2 M Tris pH 8.0. Fractionscontaining protein were then concentrated using Amicon Ultra centrifugalconcentrators. ELISA was conducted as with HEK cell supernatants. ELISAdata showing the MMP-9 dependent VEGF binding of Anti-VEGFscFv Fc ABPconstructs with the MMs 306 and 314 that were purified using a Protein Acolumn are presented in FIG. 42.

Example 12 Library Screening and Isolation of Anti-CTLA4 MMs

CTLA4 antibody masking moieties (MMs) were isolated from a combinatoriallibrary of 10¹⁰ random 15mer peptides displayed on the surface of E.coli according to the method of Bessette et al (Bessette, P. H., Rice,J. J and Daugherty, P. S. Rapid isolation of high-affinity proteinbinding peptides using bacterial display. Protein Eng. Design &Selection. 17:10,731-739, 2004). Biotinylated mouse anti-CTLA4 antibody(clone UC4 F10-11, 25 nM) was incubated with the library andantibody-bound bacteria expressing putative binding peptides weremagnetically sorted from non-binders using streptavidin-coated magneticnanobeads. Subsequent rounds of enrichment were carried out using FACS.For the initial round of FACS, bacteria were sorted using biotinylatedtarget (5 nM) and secondary labeling step with streptavidiinphycoerythrin. In subsequent rounds of FACS, sorting was performed withDylight labeled antibody and the concentration of target was reduced (1nM, then 0.1 nM) to avoid the avidity effects of the secondary labelingstep and select for the highest affinity binders. One round of MACS andthree rounds of FACS resulted in a pool of binders from which individualclones were sequenced. Relative affinity and off-rate screening ofindividual clones were performed using a ficin digested Dylight-labeledFab antibody fragment to reduce avidity effects of the bivalent antibodydue to the expression of multiple peptides on the bacterial surface. Asan additional test of target specificity, individual clones werescreened for binding in the presence of 20 uM E. Coli depleted IgG as acompetitor. Amino acid and nucleotide sequences of the 4 clones chosenfor MM optimization are shown in Table 16. These sequences willinterchangeably referred to as 187MM, 184MM, 182MM, and 175MM. MMcandidates with a range of off-rates were chosen, to determine theeffects of off-rates on MM dissociation after cleavage. An MM that didnot bind anti-CTLA4 was used as a negative control.

TABLE 16Amino acid and nucleotide sequences for MMs that mask anti-CTLA4KK187 MM M  I  L  L  C  A  A  G  R  T  W  V  E  A  C  A  N  G  R(SEQ ID NO: 63)ATGATTTTGTTGTGCGCGGCGGGTCGGACGTGGGTGGAGGCTTGCGCTAA TGGTAGG(SEQ ID NO: 64) KK184 MM A  E  R  L  C  A  W  A  G  R  F  C  G  S(SEQ ID NO: 65) GCTGAGCGGTTGTGCGCGTGGGCGGGGCGGTTCTGTGGCAGC(SEQ ID NO: 66) KK182 MM W  A  D  V  M  P  G  S  G  V  L  P  W  T  S(SEQ ID NO: 67) TGGGCGGATGTTATGCCTGGGTCGGGTGTGTTGCCGTGGACGTCG(SEQ ID NO: 68) KK175 MMS  D  G  R  M  G  S  L  E  L  C  A  L  W  G  R  F  C  G  S(SEQ ID NO: 69)AGTGATGGTCGTATGGGGAGTTTGGAGCTTTGTGCGTTGTGGGGGCGGTTCTGTGGCAGC(SEQ ID NO: 70) Negative control (does not bind anti-CTLA4)P  C  S  E  W  Q  S  M  V  Q  P  R  C  Y  Y (SEQ ID NO: 71)GCCGTGTTCTGAGTGGCAGTCGATGGTGCAGCCGCGTTGCTATTA (SEQ ID NO: 72)

Example 13 Cloning of Anti-CTLA4 scFv

Anti-CTLA4 ScFv was cloned from the HB304 hybridoma cell line (AmericanType Culture Collection) secreting UC4F10-11 hamster anti-mouse CTLA4antibody according to the method of Gilliland et al. (Gilliland L. K.,N. A. Norris, H. Marquardt, T. T. Tsu, M. S. Hayden, M. G. Neubauer, D.E. Yelton, R. S. Mittler, and J. A. Ledbetter. Rapid and reliablecloning of antibody variable regions and generation of recombinantsingle chain antibody fragments. Tissue Antigens 47:1, 1-20, 1996). Adetailed version of this protocol can be found at the Institute ofBiomedical Sciences (IBMS) at Academia Sinica in Taipei, Taiwan website.In brief, total RNA was isolated from hybridomas using the RNeasy totalRNA isolation kit (Qiagen). The primers IgK1 (gtyttrtgngtnacytcrca (SEQID NO:105)) and IgH1 (acdatyttyttrtcnacyttngt (SEQ ID NO:106))(Gilliland et al. referenced above) were used for first strand synthesisof the variable light and heavy chains, respectively. A poly G tail wasadded with terminal transferase, followed by PCR using the 5′ ANCTAILprimer (Gilliland et al. referenced above)(cgtcgatgagctctagaattcgcatgtgcaagtccgatggtcccccccccccccc: SEQ ID NO: 73)containing EcoRI, SacI and XbaI sites for both light and heavy chains(poly G tail specific) and the 3′ HBS-hIgK(cgtcatgtcgacggatccaagcttacyttccayttnacrttdatrtc: SEQ ID NO: 74) andHBS-hIgH (cgtcatgtcgacggatccaagcttrcangcnggngcnarnggrtanac: SEQ ID NO:75) derived from mouse antibody constant region sequences and containingHindIII, BamHI and SalI sites for light and heavy chain amplification,respectively (Gilliland et al. referenced above). Constructs and vectorwere digested with HindIII and SacI, ligated and transformed into E.Coli. Individual colonies were sequenced and the correct sequences forV_(L) and V_(H) (Tables 17 and 18 respectively) were confirmed bycomparison with existing mouse and hamster antibodies. The leadersequences, as described for anti-CTLA4 in the presented sequence is alsocommonly called a signal sequence or secretion leader sequence and isthe amino acid sequence that directs secretion of the antibody. Thissequence is cleaved off, by the cell, during secretion and is notincluded in the mature protein. Additionally, the same scFv cloned byTuve et al (Tuve, S. Chen, B. M., Liu, Y., Cheng, T-L., Toure, P., Sow,P. S., Feng, Q., Kiviat, N., Strauss, R., Ni, S., Li, Z., Roffler, S. R.and Lieber, A. Combination of Tumor Site-Located CTL-AssociatedAntigen-4 Blockade and Systemic Regulatory T-Cell Depletion InducesTumor Destructive Immune Responses. Cancer Res. 67:12, 5929-5939, 2007)was identical to sequences presented here.

TABLE 17 Hamster anti-mouse CTLA4 V_(L) LeaderM E S H I H V F M S L F L W V S G S C A D I M M T Q SP S S L S V S A G E K A T I S C K S S Q S L F N S NA K T N Y L N W Y L Q K P G Q S P K L L I Y Y A ST R H T G V P D R F R G S G T D F T L T I S S V Q DE D L A F Y Y C Q Q W Y D Y P Y T F G A G T K V E I K (SEQ ID NO: 76)atggaatcacatatccatgtcttcatgtccttgttcctttgggtgtctggttcctgtgcagacatcatgatgacccagtctccttcatccctgagtgtgtcagcgggagagaaagccactatcagctgcaagtccagtcagagtcttttcaacagtaacgccaaaacgaactacttgaactggtatttgcagaaaccagggcagtctcctaaactgctgatctattatgcatccactaggcatactggggtccctgatcgcttcagaggcagtggatctgggacggatttcactctcaccatcagcagtgtccaggatgaagacctggcattttattactgtcagcagtggtatgactacccatacacgttcggagctgggaccaaggtggaaatcaaa (SEQ ID NO: 77)

TABLE 18 Hamster anti mouse CTLA4 V_(H) LeaderK M R L L G L L Y L V T A L P G V L S Q I Q L Q E SG P G L V N P S Q S L S L S C S V T G Y S I T S G Y GW N W I R Q F P G Q K V E W M G F I Y Y E G S T YY N P S I K S R I S I T R D T S K N Q F F L Q V N S VT T E D T A T Y Y C A R Q T G Y F D Y W G Q G T M VT V S S (SEQ ID NO: 78)aagatgagactgttgggtcttctgtacctggtgacagcccttcctggtgtcctgtcccagatccagcttcaggagtcaggacctggcctggtgaacccctcacaatcactgtccctctcttgctctgtcactggttactccatcaccagtggttatggatggaactggatcaggcagttcccagggcagaaggtggagtggatgggattcatatattatgagggtagcacctactacaacccttccatcaagagccgcatctccatcaccagagacacatcgaagaaccagttcttcctgcaggtgaattctgtgaccactgaggacacagccacatattactgtgcgagacaaactgggtactttgattactggggccaaggaaccatggtcaccgtctcctca (SEQ ID NO: 79)

Example 14 Construction of the Anti-CTLA4 scFv with MMs and CMS

To determine the optimal orientation of the anti-CTLA4 scFv forexpression and function, primers were designed to PCR amplify thevariable light and heavy chains individually, with half of a (GGGS)₃linker at either the N- or C-terminus for a subsequent ‘splicing byoverlapping extension’ PCR (SOE-PCR; Horton, R. M., Hunt, H. D., Ho, S.N., Pullen, J. K. and Pease, L. R. (1989) Engineering hybrid geneswithout the use of restriction enzymes: gene splicing by overlapextension. Gene 77, 61-68) with either V_(H) or V_(L) at the N-terminus.An NdeI restriction site was engineered at the N-terminus to generate astart codon in frame at the beginning of the nucleotide sequence and aHis tag and stop codon were added to the C-terminus. Light and heavychains were then joined via sewing PCR using the outer primers togenerate ScFvs in both V_(H)V_(L) and V_(L)V_(H) (FIG. 43). Primers areshown below in Table 19.

TABLE 19 Primers to generate scFvs V_(H)V_(L) and V_(L)V_(H) VL for1caaggaccatagcatatggacatcatgatgacccagtct (SEQ ID NO: 80) VL linkeracttccgcctccacctgatccaccaccacctttgatttccaccttggtcc  rev1 (SEQ ID NO: 81)linker VH ggatcaggtggaggcggaagtggaggtggcggttcccagatccagcttcaggagtcaggafor2 (SEQ ID NO: 82) VH his rev2ggccggatccaagcttttagtggtgatggtgatgatgtgaggagacggtgaccatggttcc(SEQ ID NO: 83) VH for3acaaggaccatagcatatgcagatccagcttcaggagtca (SEQ ID NO: 84) VH linkeracttccgcctccacctgatccaccaccacctgaggagacggtgaccatggttcc rev3(SEQ ID NO: 85) linker VLggtggatcaggtggaggcggaagtggaggtggcggttccgacatcatgatgacccagtctcct for4(SEQ ID NO: 86) VL his rev4cggccggatccaagcttttagtggtgatggtgatgatgtttgatttccaccttggtcccagc(SEQ ID NO: 87)

Next, a set of overlapping primers were designed to add sfi and xho1sites for MM cloning followed by the MMP-9 cleavage sequence and (GGS)₂linker on the N-terminus of the ScFv constructs. These primers arepresented in Table 20 and shown schematically in FIG. 44.

TABLE 20 Primers MM and CM cloning for 1c linkergccagtctggccggtagggctcgagcggccaagtgcacatgccactgggcttcctgggtc(SEQ ID NO: 88) for 1d linker VLgccactgggcttcctgggtccgggtggaagcggcggctcagacatcatgatgacccagtc(SEQ ID NO: 89) for 1e linker VHgccactgggcttcctgggtccgggtggaagcggcggctcacagatccagcttcaggagtca(SEQ ID NO: 90) for 1a ttcaccaacaaggaccatagcatatgggccagtctggccggtagggc(SEQ ID NO: 91) VH his rev2ggccggatccaagcttttagtggtgatggtgatgatgtgaggagacggtgaccatggttcc(SEQ ID NO: 92) VH linker rev3acttccgcctccacctgatccaccaccacctgaggagacggtgaccatggttcc (SEQ ID NO: 93)

Linker containing ScFvs were PCR amplified, digested with Nde1 and EcoR1(an internal restriction site in V_(H)) and gel purified. The PCRfragments were ligated into the vectors and transformed into E. Coli.The nucleotide and amino acid sequences are presented in Table 21 andillustrated in FIG. 45.

TABLE 21 Sequence of MM linker-CM-anti-CTLA4 scFv linkerAmino acid sequence: G G S G G S G G S S G Q V H M P L G F LG P G G S G G S (SEQ ID NO: 94) Nucleotide sequence:GGCGGTTCTGGTGGCAGCGGTGGCTCGAGCGGCCAAGTGCACATGCCACTGGGCTTCCTGGGTCCGGGTGGAAGCGGCGGCTCA (SEQ ID NO: 95)

MM sequences were PCR amplified, digested at sfi1 and xho1 sites,ligated into linker anti-CTLA4 scFv constructs, transformed into E. Coliand sequenced. The complete nucleotide and amino acid sequences of theMM187-CM-TBM are shown below in Tables 22 and 23 as SEQ ID NOs: 96 and97 respectively.

TABLE 22 Amino acid sequence of MM187-anti-CTLA4 ScFv TBM:M I L L C A A G R T W V E A C A N G R G G S G G S G G S S G QV H M P L G F L G P G G S G G S Q I Q L Q E S G P G L V N P S QS L S L S C S V T G Y S I T S G Y G W N W I R Q F P G Q K V E WM G F I Y Y E G S T Y Y N P S I K S R I S I T R D T S K N Q F F LQ V N S V T T E D T A T Y Y C A R Q T G Y F D Y W G Q G T M VT V S S G G G G S G G G G S G G G G S D I M M T Q S P S S L S VS A G E K A T I S C K S S Q S L F N S N A K T N Y L N W Y L QK P G Q S P K L L I Y Y A S T R H T G V P D R F R G S G S G T DF T L T I S S V Q D E D L A F Y Y C Q Q W Y D Y P Y T F G A G TK V E I K (SEQ ID NO: 96)

TABLE 23 Nucleotide sequence of MM187-anti-CTLA4 ScFv TBM:atgattttgttgtgcgcggcgggtcggacgtgggtggaggcttgcgctaatggtaggggcggttctggtggcagcggtggctcgagcggccaagtgcacatgccactgggcttcctgggtccgggtggaagcggcggctcacagatccagcttcaggagtcaggacctggcctggtgaacccctcacaatcactgtccctctcttgctctgtcactggttactccatcaccagtggttatggatggaactggatcaggcagttcccagggcagaaggtggagtggatgggattcatatattatgagggtagcacctactacaacccttccatcaagagccgcatctccatcaccagagacacatcgaagaaccagttcttcctgcaggtgaattctgtgaccactgaggacacagccacatattactgtgcgagacaaactgggtactttgattactggggccaaggaaccatggtcaccgtctcctcaggtggtggtggatcaggtggaggcggaagtggaggtggcggttccgacatcatgatgacccagtctccttcatccctgagtgtgtcagcgggagagaaagccactatcagctgcaagtccagtcagagtcttttcaacagtaacgccaaaacgaactacttgaactggtatttgcagaaaccagggcagtctcctaaactgctgatctattatgcatccactaggcatactggggtccctgatcgcttcagaggcagtggatctgggacggatttcactctcaccatcagcagtgtccaggatgaagacctggcattttattactgtcagcagtggtatgactacccatacacgttcggagctgggaccaaggtggaaatcaaacatcatcaccatcaccactaa(SEQ ID NO: 97)

To generate MM-CM-anti-CTLA4 scFV-Fc fusions, the following primerslisted in Table 24 were designed to PCR amplify the constructs forcloning into the pfuse Fc vector via the in fusion system (Clontech).Plasmids were transformed into E. coli, and the sequence of individualclones was verified.

TABLE 24 Primers to generate MM-CM-anti-CTLA4 scFV-Fc fusionsHLCTLA4ScFv pFuse reverse tcagatctaaccatggctttgatttccaccttggtcc(SEQ ID NO: 98) LHCTLA4ScFv pFuse reversetcagatctaaccatggctgaggagacggtgaccatgg (SEQ ID NO: 99)p187CTLA4 pfuse forward cacttgtcacgaattcgatgattttgttgtgcgcggc(SEQ ID NO: 100) p182CTLA4 pfuse forwardcacttgtcacgaattcgtgggcggatgttatgcctg (SEQ ID NO: 101)p184CTLA4 pfuse forward cacttgtcacgaattcggctgagcggttgtgcgcgtg(SEQ ID NO: 102) p175CTLA4 pfuse forwardcacttgtcacgaattcgagtgatggtcgtatggggag (SEQ ID NO: 103)pnegCTLA4 pfuse forward cacttgtcacgaattcgccgtgttctgagtggcagtcg(SEQ ID NO: 104)

Example 15 Expression and Assay of Masked/MMP-9/Anti-CTLA scFv-Fc 1NHEK-293 Cells

10 ug of expression vectors for p175CTLA4pfuse, p182CTLA4pfuse,p184CTLA4pfuse, p187CTLA4pfuse, or pnegCTLA4pfuse were introduced into10⁷ HEK-293 freestyle cells (Invitrogen) by transfection usingtransfectamine 2000 as per manufacturer's protocol (Invitrogen). Thetransfected cells were incubated for an additional 72 hours. Afterincubation the conditioned media was harvested and cleared of cells anddebris by centrifugation. The conditioned media was assayed for activityby ELISA as described below.

Fifty microliters of conditioned media from HEK-293 expressingMM175-anti-CTLA4 scFv, MM182-anti-CTLA4 scFv, MM184-anti-CTLA4 scFv,MM187-anti-CTLA4 scFv, or MMneg-anti-CTLA4 scFv was added to 200 μLMMP-9 digestion buffer and incubated with or without ˜19U MMP-9 for 2hrs at 37° C. Samples were then diluted 1:1 in PBS, 4% non fat dry milk(NFDM) and assayed for binding activity by competition ELISA.

100 ul of 0.5 mg/ml solution of murine CTLA4-Fc fusion protein (R & Dsystems) in PBS was added to wells of 96 well Easy Wash plate (Corning)and incubated overnight at 4° C. Wells were then blocked for one hour atroom temperature (RT) with 100 ul of 2% non-fat dry milk (NFDM) in PBSand then washed 3× with PBS; 0.05% Tween-20 (PBST). 50 ul of conditionedmedia from cultures of transfected HEK-293 cells expressingMM175-anti-CTLA4 scFv, MM182-anti-CTLA4 scFv, MM184-anti-CTLA4 scFv,MM187-anti-CTLA4 scFv, or MMneg-anti-CTLA4 scFv that had previously beenuntreated or treated with MMP-9, were added to wells and incubated RTfor 15 minutes. Following incubation, 50 ul of PBS containing 0.5 ug/mlbiotinylated murine B71-Fc (R & D systems) was added to each well.Following a further incubation at RT of 30 minutes the wells were washed5× with 150 ul PBST. 100 ul of PBS containing 1:3000 dilution ofavidin-HRP was added and the plate incubated at RT for 45 minutes andthen washed 7× with 150 ul PBST. The ELISA was developed with 100 ul ofTMB (Pierce), stopped with 100 uL of 1N HCL and the absorbance wasmeasured at 450 nM.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of manufacturing an enzyme activatablebinding polypeptide (ABP), the method comprising: (a) culturing a cellcomprising a nucleic acid construct that encodes the ABP underconditions that lead to expression of the ABP, wherein the ABP comprisesa masking moiety (MM), a cleavable moiety (CM), and an antigen bindingdomain (ABD) that specifically binds Cytotoxic T-Lymphocyte Antigen 4(CTLA-4), (i) wherein the ABP in an uncleaved state comprises astructural arrangement from N-terminus to C-terminus as follows:MM-CM-ABD or ABD-CM-MM; (ii) wherein the MM is a peptide that inhibitsbinding of the ABD to the target, and wherein the MM comprises an aminoacid sequence selected from the group consisting of MILLCAAGRTWVEACANGR(SEQ ID NO:63), AERLCAWAGRFCGS (SEQ ID NO:65), WADVMPGSGVLPWTS (SEQ IDNO:67) and SDGRMGSLELCALWGRFCGS (SEQ ID NO:69); and (iii) wherein, theCM is positioned in the ABP such that, in an uncleaved state, the MMinterferes with specific binding of the ABD to CTLA-4, and in a cleavedstate the MM does not interfere or compete with specific binding of theABD to CTLA-4; and (b) recovering the ABP.
 2. The method of claim 1,further comprising: (c) testing the ABP for the ability to maintain anactivatable phenotype while in soluble form.
 3. The method of claim 1,wherein the MM is a peptide of no more than about 40 amino acids inlength.
 4. The method of claim 1, wherein the ABD comprises a Fabfragment, a scFv or a single chain antibody (SCAB).
 5. The method ofclaim 1, wherein the CM is a polypeptide that functions as a substratefor a protease that is co-localized in a tissue with the target, whereinthe protease cleaves the CM in the ABP when the ABP is exposed to theprotease.
 6. The method of claim 1, wherein the CM is a polypeptide ofup to 15 amino acids in length.
 7. The method of claim 1, wherein the CMof the ABP in an uncleaved state is coupled to the N-terminus of theABD.
 8. The method of claim 7, wherein the CM of the ABP in an uncleavedstate is coupled to the N-terminus of a V_(L) chain of the ABD.
 9. Themethod of claim 1, wherein the CM of the ABP in an uncleaved state iscoupled to the C-terminus of the ABD.
 10. The method of claim 1, whereinthe ABD is from ipilimumab or tremelimumab.
 11. The method of claim 1,wherein the CM is a substrate for an enzyme selected from the groupconsisting of MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA,PSMA, CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS,Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6,Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12,Caspase-13, Caspase-14, and TACE.
 12. The method of claim 1, wherein theCM is a substrate for an enzyme selected from the group consisting of anMMP and a CATHEPSIN.
 13. The method of claim 1, wherein the ABPcomprises a linker peptide, wherein the linker peptide is positionedbetween the MM and the CM.
 14. The method of claim 1, wherein the ABPcomprises a linker peptide, wherein the linker peptide is positionedbetween the ABD and the CM.
 15. The method of claim 1, wherein the ABPcomprises a first linker peptide (L₁) and a second linker peptide (L₂),wherein the first linker peptide is positioned between the MM and the CMand the second linker peptide is positioned between the ABD and the CM.16. The method of claim 15, wherein each of L₁ and L₂ is a peptide ofabout 1 to 20 amino acids in length, and wherein each of L₁ and L₂ neednot be the same linker.
 17. The method of claim 15, wherein one or bothof L₁ and L₂ comprises a glycine-serine polymer.
 18. The method of claim15, wherein at least one of L₁ and L₂ comprises an amino acid sequenceselected from the group consisting of (GS)_(n), (GSGGS)_(n)(SEQ ID NO:1)and (GGGS)_(n)(SEQ ID NO:2), where n is an integer of at least one. 19.The method of claim 15, wherein at least one of L₁ and L₂ comprises anamino acid sequence having the formula (GGS)_(n), where n is an integerof at least one.
 20. The method of claim 15, wherein at least one of L₁and L₂ comprises an amino acid sequence selected from the groupconsisting of Gly-Gly-Ser-Gly (SEQ ID NO:3), Gly-Gly-Ser-Gly-Gly (SEQ IDNO:4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:5), Gly-Ser-Gly-Gly-Gly (SEQ IDNO:6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO:7), and Gly-Ser-Ser-Ser-Gly (SEQID NO:8).
 21. A method of manufacturing an enzyme activatable bindingpolypeptide (ABP), the method comprising: (a) providing a masking moiety(MM), a cleavable moiety (CM), and an antibody or an antigen bindingfragment thereof (ABD) that specifically binds Cytotoxic T-LymphocyteAntigen 4 (CTLA-4), wherein the MM is a peptide that inhibits binding ofthe ABD to CTLA-4, and wherein the MM comprises an amino acid sequenceselected from the group consisting of MILLCAAGRTWVEACANGR (SEQ IDNO:63), AERLCAWAGRFCGS (SEQ ID NO:65), WADVMPGSGVLPWTS (SEQ ID NO:67)and SDGRMGSLELCALWGRFCGS (SEQ ID NO:69); and (b) coupling the MM to theCM and coupling the ABD to the CM to produce an ABP, wherein: (i) theABP in an uncleaved state comprises a structural arrangement fromN-terminus to C-terminus as follows: MM-CM-ABD or ABD-CM-MM; and (ii)the CM is positioned in the ABP such that, in an uncleaved state, the MMinterferes with specific binding of the ABD to CTLA-4 and in a cleavedstate the MM does not interfere or compete with specific binding of theABD to CTLA-4.
 22. The method of claim 21, further comprising: (c)testing the ABP for the ability to maintain an activatable phenotypewhile in soluble form.
 23. The method of claim 21, wherein the MM is apeptide of no more than about 40 amino acids in length.
 24. The methodof claim 21, wherein the ABP is manufactured by culturing a cellcomprising a nucleic acid construct that encodes the ABP underconditions that lead to expression of the ABP.
 25. The method of claim21, wherein the ABD comprises a Fab fragment, a scFv or a single chainantibody (SCAB).
 26. The method of claim 21, wherein the CM is apolypeptide that functions as a substrate for a protease that isco-localized in a tissue with the target, wherein the protease cleavesthe CM in the ABP when the ABP is exposed to the protease.
 27. Themethod of claim 21, wherein the CM is a polypeptide of up to 15 aminoacids in length.
 28. The method of claim 21, wherein the CM of the ABPin an uncleaved state is coupled to the N-terminus of the ABD.
 29. Themethod of claim 28, wherein the CM of the ABP in an uncleaved state iscoupled to the N-terminus of a V_(L) chain of the ABD.
 30. The method ofclaim 21, wherein the CM of the ABP in an uncleaved state is coupled tothe C-terminus of the ABD.
 31. The method of claim 21, wherein the ABDis from ipilimumab or tremelimumab.
 32. The method of claim 21, whereinthe CM is a substrate for an enzyme selected from the group consistingof MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-14, plasmin, PSA, PSMA,CATHEPSIN D, CATHEPSIN K, CATHEPSIN S, ADAM10, ADAM12, ADAMTS,Caspase-1, Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6,Caspase-7, Caspase-8, Caspase-9, Caspase-10, Caspase-11, Caspase-12,Caspase-13, Caspase-14, and TACE.
 33. The method of claim 21, whereinthe CM is a substrate for an enzyme selected from the group consistingof an MMP and a CATHEPSIN.
 34. The method of claim 21, wherein the ABPcomprises a linker peptide, wherein the linker peptide is positionedbetween the MM and the CM.
 35. The method of claim 21, wherein the ABPcomprises a linker peptide, wherein the linker peptide is positionedbetween the ABD and the CM.
 36. The method of claim 21, wherein the ABPcomprises a first linker peptide (L₁) and a second linker peptide (L₂),wherein the first linker peptide is positioned between the MM and the CMand the second linker peptide is positioned between the ABD and the CM.37. The method of claim 36, wherein each of L₁ and L₂ is a peptide ofabout 1 to 20 amino acids in length, and wherein each of L₁ and L₂ neednot be the same linker.
 38. The method of claim 36, wherein one or bothof L₁ and L₂ comprises a glycine-serine polymer.
 39. The method of claim36, wherein at least one of Ll and L2 comprises an amino acid sequenceselected from the group consisting of (GS)_(n), (GSGGS)_(n)(SEQ ID NO:1)and (GGGS)_(n) (SEQ ID NO:2), where n is an integer of at least one. 40.The method of claim 36, wherein at least one of L₁ and L₂ comprises anamino acid sequence having the formula (GGS)_(n), where n is an integerof at least one.
 41. The method of claim 36, wherein at least one of L₁and L₂ comprises an amino acid sequence selected from the groupconsisting of Gly-Gly-Ser-Gly (SEQ ID NO:3), Gly-Gly-Ser-Gly-Gly (SEQ IDNO:4), Gly-Ser-Gly-Ser-Gly (SEQ ID NO:5), Gly-Ser-Gly-Gly-Gly (SEQ IDNO:6), Gly-Gly-Gly-Ser-Gly (SEQ ID NO:7), and Gly-Ser-Ser-Ser-Gly (SEQID NO:8).